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Triple-negative breast cancer (TNBC) patients with upregulated Wnt/β-catenin signaling often have poor clinical prognoses. During pathological examinations of breast cancer sections stained for β-catenin, we made the serendipitous observation that relative to non-TNBC, specimens from TNBC patients have a greater abundance of nucleoli. There was a remarkable direct relationship between nuclear β-catenin and greater numbers of nucleoli in TNBC tissues. These surprising observations spurred our investigations to decipher the differential functional relevance of the nucleolus in TNBC versus non-TNBC cells. Comparative nucleolar proteomics revealed that the majority of the nucleolar proteins in TNBC cells were potential targets of β-catenin signaling. Next, we undertook an analysis of the nucleolar proteome in TNBC cells in response to β-catenin inhibition. This effort revealed that a vital component of pre-rRNA processing, LAS1 like ribosome biogenesis factor (LAS1L) was significantly decreased in the nucleoli of β-catenin inhibited TNBC cells. Here we demonstrate that LAS1L protein expression is significantly elevated in TNBC patients, and it functionally is important for mammary tumor growth in xenograft models and enables invasive attributes. Our observations highlight a novel function for β-catenin in orchestrating nucleolar activity in TNBCs.

Background Poly (ADP-ribose)-polymerase inhibitors (PARPi) have been approved for cancer patients with germline BRCA1/2 (gBRCA1/2) mutations, and efforts to expand the utility of PARPi beyond BRCA1/2 are ongoing. In preclinical models of triple-negative breast cancer (TNBC) with intact DNA repair, we have previously shown an induced synthetic lethality with combined EGFR inhibition and PARPi. Here, we report the safety and clinical activity of lapatinib and veliparib in patients with metastatic TNBC. Methods A first-in-human, pilot study of lapatinib and veliparib was conducted in metastatic TNBC (NCT02158507). The primary endpoint was safety and tolerability. Secondary endpoints were objective response rates and pharmacokinetic evaluation. Gene expression analysis of pre-treatment tumor biopsies was performed. Key eligibility included TNBC patients with measurable disease and prior anthracycline-based and taxane chemotherapy. Patients with gBRCA1/2 mutations were excluded. Results Twenty patients were enrolled, of which 17 were evaluable for response. The median number of prior therapies in the metastatic setting was 1 (range 0–2). Fifty percent of patients were Caucasian, 45% African–American, and 5% Hispanic. Of evaluable patients, 4 demonstrated a partial response and 2 had stable disease. There were no dose-limiting toxicities. Most AEs were limited to grade 1 or 2 and no drug–drug interactions noted. Exploratory gene expression analysis suggested baseline DNA repair pathway score was lower and baseline immunogenicity was higher in the responders compared to non-responders. Conclusions Lapatinib plus veliparib therapy has a manageable safety profile and promising antitumor activity in advanced TNBC. Further investigation of dual therapy with EGFR inhibition and PARP inhibition is needed. Trial registration ClinicalTrials.gov, NCT02158507. Registered on 12 September 2014

The expression of Merlin tumor suppressor protein encoded by Neurofibromin 2 (NF2) gene is remarkably decreased in metastatic breast cancer tissues. In order to recapitulate clinical evidence, we generated a unique, conditional Nf2‐knockout (Nf2−/−) mouse mammary tumor model. Merlin‐deficient breast tumor cells and Nf2−/− mouse embryonic fibroblasts (MEFs) displayed a robustly invasive phenotype. Moreover, Nf2−/− MEFs presented with notable alterations in redox management networks, implicating a role for Merlin in redox homeostasis. This programmatic alteration resonated with pathways that emerged from breast tumor cells engineered for Merlin deficiency. Further investigations revealed that NF2‐silenced cells supported reduced activity of the Nuclear factor, erythroid 2 like 2 antioxidant transcription factor, concomitant with elevated expression of NADPH oxidase enzymes. Importantly, mammary‐specific Nf2−/− in an Mouse mammary tumor virus Neu + murine breast cancer model demonstrated accelerated mammary carcinogenesis in vivo. Tumor‐derived primary organoids and cell lines were characteristically invasive with evidence of a dysregulated cellular redox management system. As such, Merlin deficiency programmatically influences redox imbalance that orchestrates malignant attributes of mammary/breast cancer., Merlin‐deficient cells have an under‐functioning Nuclear factor, erythroid 2 like 2 antioxidant system, as well as a NOX‐enabled redox production system in hyperdrive. This leads to intensified oxidative stress that converges upon unrestricted cell growth and intensification of malignant attributes. Thus, Merlin deficiency disables the cellular redox management system. The findings uncover a novel mechanism by which Merlin intersects with redox homeostasis in restricting malignant characteristics.

High attrition rates in tuberculosis (TB) drug development have been largely attributed to safety, which is likely due to the use of endpoint assays measuring cell viability to detect drug cytotoxicity. In drug development for cancer, metabolic, and neurological disorders and for antibiotics, cytotoxicity is increasingly being assessed using extracellular flux (XF) analysis, which measures cellular bioenergetic metabolism in real time. Here, we adopt the XF platform to investigate the cytotoxicity of drugs currently used in TB treatment on the bioenergetic metabolism of HepG2 cells, THP-1 macrophages, and human monocyte-derived macrophages (hMDMs). We found that the XF analysis reveals earlier drug-induced effects on the cells' bioenergetic metabolism prior to cell death, measured by conventional viability assays. Furthermore, each cell type has a distinct response to drug treatment, suggesting that more than one cell type should be considered to examine cytotoxicity in TB drug development. Interestingly, chemically unrelated drugs with different modes of action on Mycobacterium tuberculosis have similar effects on the bioenergetic parameters of the cells, thus discouraging the prediction of potential cytotoxicity based on chemical structure and mode of action of new chemical entities. The clustering of the drug-induced effects on the hMDM bioenergetic parameters are reflected in the clustering of the effects of the drugs on cytokine production in hMDMs, demonstrating concurrence between the effects of the drugs on the metabolism and functioning of the macrophages. These findings can be used as a benchmark to establish XF analysis as a new tool to assay cytotoxicity in TB drug development.

Metastatic breast cancer causes most breast cancer-associated deaths, especially in triple negative breast cancers (TNBC). The metastatic drivers of TNBCs are still poorly understood, and effective treatment non-existent. Here we reveal that the presence of Aurora-A Kinase (AURKA) in the nucleus and metastatic dissemination are molecularly connected through HIF1 (Hypoxia-Inducible Factor-1) signaling. Nuclear AURKA activates transcription of "hypoxia-induced genes" under normoxic conditions (pseudohypoxia) and without upregulation of oxygen-sensitive HIF1A subunit. We uncover that AURKA preferentially binds to HIF1B and co-localizes with the HIF complex on DNA. The mass-spectrometry analysis of the AURKA complex further confirmed the presence of CBP and p300 along with other TFIIB/RNApol II components. Importantly, the expression of multiple HIF-dependent genes induced by nuclear AURKA (N-AURKA), including migration/invasion, survival/death, and stemness, promote early cancer dissemination. These results indicate that nuclear, but not cytoplasmic, AURKA is a novel driver of early metastasis. Analysis of clinical tumor specimens revealed a correlation between N-AURKA presence and decreased patient survival. Our results establish a mechanistic link between two critical pathways in cancer metastasis, identifying nuclear AURKA as a crucial upstream regulator of the HIF1 transcription complex and a target for anti-metastatic therapy.

Gemcitabine is used to treat pancreatic cancer (PC), but is not curative. We sought to determine whether gemcitabine + a BET bromodomain inhibitor was superior to gemcitabine, and identify proteins that may contribute to the efficacy of this combination. This study was based on observations that cell cycle dysregulation and DNA damage augment the efficacy of gemcitabine. BET inhibitors arrest cells in G1 and allow increases in DNA damage, likely due to inhibition of expression of DNA repair proteins Ku80 and RAD51. BET inhibitors (JQ1 or I-BET762) + gemcitabine were synergistic in vitro, in Panc1, MiaPaCa2 and Su86 PC cell lines. JQ1 + gemcitabine was more effective in vivo than either drug alone in patient-derived xenograft models (P &lt, 0.01). Increases in the apoptosis marker cleaved caspase 3 and DNA damage marker γH2AX paralleled antitumor efficacy. Notably, RNA-seq data showed that JQ1 + gemcitabine selectively inhibited HMGCS2 and APOC1 ~6-fold, compared to controls. These proteins contribute to cholesterol biosynthesis and lipid metabolism, and their overexpression supports tumor cell proliferation. IPA data indicated that JQ1 + gemcitabine selectively inhibited the LXR/RXR activation pathway, suggesting the hypothesis that this inhibition may contribute to the observed in vivo efficacy of JQ1 + gemcitabine.

Background: Administration of amplitude modulated 27·12 MHz radiofrequency electromagnetic fields (AM RF EMF) by means of a spoon-shaped applicator placed on the patient's tongue is a newly approved treatment for advanced hepatocellular carcinoma (HCC). The mechanism of action of tumour-specific AM RF EMF is largely unknown. Methods: Whole body and organ-specific human dosimetry analyses were performed. Mice carrying human HCC xenografts were exposed to AM RF EMF using a small animal AM RF EMF exposure system replicating human dosimetry and exposure time. We performed histological analysis of tumours following exposure to AM RF EMF. Using an agnostic genomic approach, we characterized the mechanism of action of AM RF EMF. Findings: Intrabuccal administration results in systemic delivery of athermal AM RF EMF from head to toe at levels lower than those generated by cell phones held close to the body. Tumour shrinkage results from differentiation of HCC cells into quiescent cells with spindle morphology. AM RF EMF targeted antiproliferative effects and cancer stem cell inhibiting effects are mediated by Ca2+ influx through Cav3·2 T-type voltage-gated calcium channels (CACNA1H) resulting in increased intracellular calcium concentration within HCC cells only. Interpretation: Intrabuccally-administered AM RF EMF is a systemic therapy that selectively block the growth of HCC cells. AM RF EMF pronounced inhibitory effects on cancer stem cells may explain the exceptionally long responses observed in several patients with advanced HCC. Fund: Research reported in this publication was supported by the National Cancer Institute's Cancer Centre Support Grant award number P30CA012197 issued to the Wake Forest Baptist Comprehensive Cancer Centre (BP) and by funds from the Charles L. Spurr Professorship Fund (BP). DWG is supported by R01 AA016852 and P50 AA026117. Keywords: Advanced hepatocellular carcinoma, T-type voltage gated calcium channels, Calcium influx, Cav 3·2, CACNA1H, Amplitude-modulated, Radiofrequency, Electromagnetic fields, AM RF EMF

The chromosome 8q24.21 locus, which contains the proto-oncogene c-MYC, long non-coding RNA PVT1, and microRNAs (miRs), is the most commonly amplified region in human prostate cancer. A long-range interaction of genetic variants with c-MYC or long non-coding PVT1 at this locus contributes to the genetic risk of prostate cancer. At this locus is a cluster of genes for six miRs (miR-1204, -1205, -1206, -1207-3p, -1207-5p, and -1208), but their functional role remains elusive. Here the copy numbers and expression levels of miRs-1204–1208 were investigated using quantitative PCR for prostate cancer cell lines and primary tumors. The data revealed that copy numbers and expression of miR-1205 were increased in both castration-resistant prostate cancer cell lines and in primary tumors. In castration-resistant prostate cancer specimens, the copy number at the miR-1205 locus correlated with the expression of miR-1205. Furthermore, functional analysis with an miR-1205 mimic, an miR-1205 inhibitor, and CRISPR/Cas9 knockout revealed that, in human prostate cancer cells, miR-1205 promoted cell proliferation and cell cycle progression and inhibited hydrogen peroxide-induced apoptosis. In these cells, miR-1205 downregulated the expression of the Egl-9 family hypoxia inducible factor 3(EGLN3) gene and targeted a site in its 3′-untranslated region to downregulate its transcriptional activity. Thus, by targeting EGLN3, miR-1205 has an oncogenic role and may contribute to the genetic risk of castration-resistant prostate cancer.

Molecular dynamics of developmental processes are repurposed by cancer cells to support cancer initiation and progression. Disruption of the delicate balance between cellular differentiation and plasticity during mammary development leads to breast cancer initiation and metastatic progression. STAT5A is essential for differentiation of secretory mammary alveolar epithelium. Active STAT5A characterizes breast cancer patients for favorable prognosis. N-Myc and STAT Interactor protein (NMI) was initially discovered as a protein that interacts with various STATs; however, the relevance of these interactions to normal mammary development and cancer was not known. We observe that NMI protein is expressed in the mammary ductal epithelium at the onset of puberty and is induced in pregnancy. NMI protein is decreased in 70% of patient specimens with metastatic breast cancer compared to primary tumors. Here we present our finding that NMI and STAT5A cooperatively mediate normal mammary development. Loss of NMI in vivo caused a decrease in STAT5A activity in normal mammary epithelial as well as breast cancer cells. Analysis of STAT5A mammary specific controlled genetic program in the context of NMI knockout revealed ISG20 (interferon stimulated exonuclease gene 20, a protein involved in rRNA biogenesis) as an unfailing negatively regulated target. Role of ISG20 has never been described in metastatic process of mammary tumors. We observed that overexpression of ISG20 is increased in metastases compared to matched primary breast tumor tissues. Our observations reveal that NMI-STAT5A mediated signaling keeps a check on ISG20 expression via miR-17–92 cluster. We show that uncontrolled ISG20 expression drives tumor progression and metastasis.

High attrition rates in tuberculosis (TB) drug development have been largely attributed to safety, which is likely due to the use of endpoint assays measuring cell viability to detect drug cytotoxicity. In drug development of cancer, metabolic and neurological disorders, and antibiotics, cytotoxicity is increasingly being assessed using extracellular flux (XF) analysis, which measures cellular bioenergetic metabolism in real-time. Here, we adopt the XF platform to investigate the cytotoxicity of drugs currently used in TB treatment on the bioenergetic metabolism of HepG2 cells, THP-1 macrophages, and human monocyte derived macrophages (hMDM). We found that the XF analysis reveals earlier drug-induced effects on the cells’ bioenergetic metabolism prior to cell death, measured by conventional viability assays. Furthermore, each cell type has a distinct response to drug treatment, suggesting that more than one cell type should be considered to examine cytotoxicity in TB drug development. Interestingly, chemically unrelated drugs with different modes of action on Mycobacterium tuberculosis have similar effects on the bioenergetic parameters of the cells, thus, discouraging the prediction of potential cytotoxicity based on chemical structure and mode of action of new chemical entities. The clustering of the drug-induced effects on the hMDM bioenergetic parameters are reflected in the clustering of the effects of the drugs on cytokine production in hMDMs, demonstrating concurrence between the effects of the drugs on the metabolism and functioning of the macrophages. These findings can be used as a benchmark to establish XF analysis as a new tool to assay cytotoxicity in TB drug development.

The highly lethal brain cancer glioblastoma undergoes dynamic changes in molecular profile and cellular phenotype throughout tumor core establishment and in primary-to-recurrent tumor progression. These dynamic changes allow glioblastoma tumors to escape from multimodal therapies, resulting in patient lethality. Here, we identified the emergence of dependence on NEK2-mediated EZH2 signaling, specifically in therapy-resistant tumor core-located glioblastoma cells. In patient-derived glioblastoma core models, NEK2 was required for in vivo tumor initiation, propagation, and radio-resistance. Mechanistically, in glioblastoma core cells, NEK2 binds with EZH2 to prevent its proteasome-mediated degradation in a kinase-dependent manner. Clinically, NEK2 expression is elevated in recurrent tumors after therapeutic failure as opposed to their matched primary untreated cases, and its high expression is indicative of worse prognosis. For therapeutic development, we designed a novel NEK2 kinase inhibitor CMP3a, which effectively attenuated growth of murine glioblastoma models and exhibited a synergistic effect with radiation therapy. Collectively, the emerging NEK2-EZH2 signaling axis is critical in glioblastoma, particularly within the tumor core, and the small molecule inhibitor CMP3a for NEK2 is a potential novel therapeutic agent for glioblastoma.

CHD7 encodes an ATP-dependent chromatin remodeling factor. Mutation of this gene causes multiple developmental disorders, including CHARGE (Coloboma of the eye, Heart defects, Atresia of the choanae, Retardation of growth/development, Genital abnormalities, and Ear anomalies) syndrome, in which conotruncal anomalies are the most prevalent form of heart defects. How CHD7 regulates conotruncal development remains unclear. In this study, we establish that deletion of Chd7 in neural crest cells (NCCs) causes severe conotruncal defects and perinatal lethality, thus providing mouse genetic evidence demonstrating that CHD7 cell-autonomously regulates cardiac NCC development, thereby clarifying a long-standing controversy in the literature. Using transcriptomic analyses, we show that CHD7 fine-tunes the expression of a gene network that is critical for cardiac NCC development. To gain further molecular insights into gene regulation by CHD7, we performed a protein-protein interaction screen by incubating recombinant CHD7 on a protein array. We find that CHD7 directly interacts with several developmental disorder-mutated proteins including WDR5, a core component of H3K4 methyltransferase complexes. This direct interaction suggested that CHD7 may recruit histone-modifying enzymes to target loci independently of its remodeling functions. We therefore generated a mouse model that harbors an ATPase-deficient allele and demonstrates that mutant CHD7 retains the ability to recruit H3K4 methyltransferase activity to its targets. Thus, our data uncover that CHD7 regulates cardiovascular development through ATP-dependent and -independent activities, shedding light on the etiology of CHD7-related congenital disorders. Importantly, our data also imply that patients carrying a premature stop codon versus missense mutations will likely display different molecular alterations; these patients might therefore require personalized therapeutic interventions.

The mechanisms by which HIV increases susceptibility to tuberculosis and other respiratory infections are incompletely understood. We used transcriptomics of paired whole bronchoalveolar lavage cells (BLCs) and peripheral blood mononuclear cells to compare the effect of HIV at the lung mucosal surface and in peripheral blood. The majority of HIV-induced differentially expressed genes (DEGs) were specific to either the peripheral or lung mucosa compartments (1,307/1,404, 93%). Type I interferon signaling was the dominant signature of DEGs in HIV-positive blood but not in HIV-positive BLCs. DEGs in the HIV-positive BLCs were significantly enriched for infiltration with cytotoxic CD8+ T cells. Higher expression of type 1 interferon transcripts in peripheral CD8+ T cells and representative transcripts and proteins in BLCs-derived CD8+ T cells during HIV infection, including IFNG (IFN-gamma), GZMB (Granzyme B), and PDCD1 (PD-1), was confirmed by cell-subset specific transcriptional analysis and flow cytometry. Thus, we report that a whole transcriptomic approach revealed qualitatively distinct effects of HIV in blood and bronchoalveolar compartments. Further work exploring the impact of distinct type I interferon programs and functional features of CD8+ T cells infiltrating the lung mucosa during HIV infection may provide novel insights into HIV-induced susceptibility to respiratory pathogens.

Congenital cytomegalovirus (cCMV) infection is the most common congenital infection and a leading cause of long-term neurological and sensory sequelae, the most common being sensorineural hearing loss (SNHL). Despite extensive research, clinical or laboratory markers to identify CMV infected children with increased risk for disease have not been identified. This study utilizes viral whole-genome next generation-sequencing (NGS) of specimens from congenitally infected infants to explore viral diversity and specific viral variants that may be associated with symptomatic infection and SNHL.CMV DNA from urine specimens of 30 infants (17 asymptomatic, 13 symptomatic) was target enriched and next generation sequenced resulting in 93% coverage of the CMV genome allowing analysis of viral diversity.Variant frequency distribution was compared between children with symptomatic and asymptomatic cCMV and those with (n = 13) and without (n = 17) hearing loss. The CMV genes UL48A, UL88, US19 and US22 were found to have an increase in nucleotide diversity in symptomatic children; while UL57, UL20, UL104, US14, UL115, and UL35 had an increase in diversity in children with hearing loss. An analysis of single variant differences between symptomatic and asymptomatic children found UL55 to have the highest number, while the most variants associated with SNHL were in the RL11 gene family. In asymptomatic infants with SNHL, mutations were observed more frequently in UL33 and UL20.CMV genomes from infected newborns can be mapped to 93% of the genome at a depth allowing accurate and reproducible analysis of polymorphisms for variant and gene discovery that may be linked to symptomatic and hearing loss outcomes.

Pediatric high-grade brain tumors and adult glioblastoma are associated with significant morbidity and mortality. Oncolytic herpes simplex virus-1 (oHSV) is a promising approach to target brain tumors; oHSV G207 and M032 (encodes human interleukin-12) are currently in phase I clinical trials in children with malignant supratentorial brain tumors and adults with glioblastoma, respectively. We sought to compare the sensitivity of patient-derived pediatric malignant brain tumor and adult glioblastoma xenografts to these clinically-relevant oHSV. In so doing we found that pediatric brain tumors were more sensitive to the viruses and expressed significantly more nectin-1 (CD111) than adult glioblastoma. Pediatric embryonal and glial tumors were 74-fold and 14-fold more sensitive to M002 and 16-fold and 6-fold more sensitive to G207 than adult glioblastoma, respectively. Of note, pediatric embryonal tumors were more sensitive than glial tumors. Differences in sensitivity may be due in part to nectin-1 expression, which predicted responses to the viruses. Treatment with oHSV resulted in prolonged survival in both pediatric and adult intracranial patient-dervied tumor xenograft models. Our results suggest that pediatric brain tumors are ideal targets for oHSV and that brain tumor expression of nectin-1 may be a useful biomarker to predict patient response to oHSV.

The process of organ development requires a delicate balance between cellular plasticity and differentiation. This balance is disrupted in cancer initiation and progression. N-Myc and STAT Interactor (NMI: human or Nmi: murine) has emerged as a relevant player in the etiology of breast cancer. However, a fundamental understanding of its relevance to normal mammary biology is lacking. To gain insight into its normal function in mammary gland, we generated a mammary specific Nmi knockout mouse model. We observed that Nmi protein expression is induced in mammary epithelium at the onset of pregnancy, in luminal cells and persists throughout lactation. Nmi knockout results in a precocious alveolar phenotype. These alveoli exhibit an extensive presence of nuclear β-catenin and enhanced Wnt/β-catenin signaling. The Nmi-knockout pubertal ductal tree shows enhanced invasion of the mammary fatpad and increased terminal end bud numbers. Tumors from Nmi null mammary epithelium show a significant enrichment of poorly differentiated cells with elevated stem/progenitor markers, active Wnt/β-catenin signaling, highly invasive morphology as well as, increased number of distant metastases. Our study demonstrates that Nmi has a distinct role in the differentiation process of mammary luminal epithelial cell compartment and developmental aberrations resulting from Nmi absence contribute to metastasis and demonstrates that aberration in normal developmental program can lead to metastatic disease, highlighting the contribution and importance of luminal progenitor cells in driving metastatic disease.

Purpose: A hallmark of neoplasia is increased ribosome biogenesis, and targeting this process with RNA polymerase I (Pol I) inhibitors has shown some efficacy. We examined the contribution and potential targeting of ribosomal machinery in chemotherapy-resistant and -sensitive models of ovarian cancer. Experimental Design: Pol I machinery expression was examined, and subsequently targeted with the Pol I inhibitor CX-5461, in ovarian cancer cell lines, an immortalized surface epithelial line, and patient-derived xenograft (PDX) models with and without chemotherapy. Effects on viability, Pol I occupancy of rDNA, ribosomal content, and chemosensitivity were examined. Results: In PDX models, ribosomal machinery components were increased in chemotherapy-treated tumors compared with controls. Thirteen cell lines were sensitive to CX-5461, with IC50s 25 nmol/L–2 μmol/L. Interestingly, two chemoresistant lines were 10.5- and 5.5-fold more sensitive than parental lines. CX-5461 induced DNA damage checkpoint activation and G2–M arrest with increased γH2AX staining. Chemoresistant cells had 2- to 4-fold increased rDNA Pol I occupancy and increased rRNA synthesis, despite having slower proliferation rates, whereas ribosome abundance and translational efficiency were not impaired. In five PDX models treated with CX-5461, one showed a complete response, one a 55% reduction in tumor volume, and one maintained stable disease for 45 days. Conclusions: Pol I inhibition with CX-5461 shows high activity in ovarian cancer cell lines and PDX models, with an enhanced effect on chemoresistant cells. Effects occur independent of proliferation rates or dormancy. This represents a novel therapeutic approach that may have preferential activity in chemoresistant populations. Clin Cancer Res; 23(21); 6529–40. ©2017 AACR.

Members of the microRNA (miR)-200 family, which are involved in tumor metastasis, have potential as cancer biomarkers, but their regulatory mechanisms remain elusive. We investigated FOXP3-inducible breast cancer cells, Foxp3 heterozygous Scurfy mutant (Foxp3 sf/+ ) female mice, and patients with breast cancer for characterization of the formation and regulation of the miR-200 family in breast cancer cells and circulation. Participants (259), including patients with breast cancer or benign breast tumors, members of breast cancer families, and healthy controls, were assessed for tumor and circulating levels of the miR-200 family. First, we identified a FOXP3-KAT2B-miR-200c/141 axis in breast cancer cells. Second, aging Foxp3 sf/+ female mice developed spontaneous breast cancers and lung metastases. Levels of miR-200c and miR-141 were lower in Foxp3 sf/+ tumor cells than in normal breast epithelial cells, but plasma levels of miR-200c and miR-141 in the Foxp3 sf/+ mice increased during tumor progression and metastasis. Third, in patients with breast cancer, the levels of miR-200c and 141 were lower in FOXP3 low relative to those with FOXP3 high breast cancer cells, especially in late-stage and metastatic cancer cells. The levels of miR-200c and miR-141 were higher in plasma from patients with metastatic breast cancer than in plasma from those with localized breast cancer, with benign breast tumors, with a family history of breast cancer, or from healthy controls. Finally, in Foxp3 sf/+ mice, plasma miR-200c and miR-141 appeared to be released from tumor cells. miR-200c and miR-141 are regulated by a FOXP3-KAT2B axis in breast cancer cells, and circulating levels of miR-200c and miR-141 are potential biomarkers for early detection of breast cancer metastases.

The low-density lipoprotein receptor-related protein 6 (LRP6) is an essential Wnt co-receptor of the Wnt/β-catenin signaling pathway. Although studies have shown an increased expression of LRP6 in several types of cancer, its function in tumor development and progression remains to be elucidated. We herein demonstrated that LRP6 expression is up-regulated in human triple negative breast cancer (TNBC) patients and human TNBC cell lines, and that knockdown of LRP6 expression and treatment of recombinant Mesd protein (a specific inhibitor of LRP6) significantly decreased cell migration and invasion of TNBC MDA-MB-231 and BT549 cells. Interestingly, the effects of LRP6 knockdown and Mesd treatment on TNBC cell migration and invasion were more prominent than on TNBC cell proliferation/viability. Mechanistically, LRP6 knockdown and Mesd treatment inhibited Wnt/β-catenin signaling and decreased the expression of S100A4, a mediator of cancer metastasis and a specific target of Wnt/β-catenin signaling, in TNBC cells. Together, our data suggest that LRP6 promotes TNBC cell migration and invasion by regulating the expression and function of S100A4 via the Wnt/β-catenin signaling pathway. J. Cell. Biochem. 118: 2968-2976, 2017. © 2017 Wiley Periodicals, Inc.

The introduction of targeted therapies has caused a paradigm shift in the treatment of metastatic clear cell (cc)-renal cell carcinoma (RCC). We hypothesized that determining differential kinase activity between primary and metastatic tumor sites may identify critical drivers of progression and relevant therapeutic targets in metastatic disease. Kinomic profiling was performed on primary tumor and metastatic tumor deposits utilizing a peptide substrate microarray to detect relative tyrosine phosphorylation activity. Pharmacologic and genetic loss of function experiments were used to assess the biologic significance of the top scoring kinase on in vitro and in vivo tumor phenotypes. Kinomics identified 7 peptides with increased tyrosine phosphorylation in metastases that were significantly altered (p

// Eddy S. Yang 1, * , Christopher D. Willey 1, * , Amitkumar Mehta 2, * , Michael R. Crowley 3 , David K. Crossman 3 , Dongquan Chen 4 , Joshua C. Anderson 1 , Gurudatta Naik 2 , Deborah L. Della Manna 1 , Tiffiny S. Cooper 1 , Guru Sonpavde 2 1 Department of Radiation Oncology, University of Alabama, Birmingham (UAB), Birmingham, Alabama, USA 2 Department of Medicine, Section of Oncology, UAB School of Medicine, Birmingham, Alabama, USA 3 Department of Genetics, UAB School of Medicine, Birmingham, Alabama, USA 4 Department of Medicine, Division of Preventive Medicine, Biostatistics and Bioinformatics Shared Facility, UAB Comprehensive Cancer Center, Birmingham, Alabama, USA * These authors have equal credit Correspondence to: Guru Sonpavde, email: gsonpavde@uabmc.edu Keywords: penile squamous cell carcinoma, kinases, DNA, RNA, protein Received: August 23, 2016 Accepted: January 23, 2017 Published: February 21, 2017 ABSTRACT Penile squamous cell carcinoma (PSCC) is an orphan malignancy with poorly understood biology and suboptimal systemic therapy. Given that kinases may be drivers and readily actionable, we performed comprehensive multiplatform analysis of kinases in PSCC tumor and normal tissue. Fresh frozen tumors were collected from 11 patients with PSCC. After macrodissection to demarcate tumor from normal tissue, the samples underwent multiplatform analysis of kinases. Next Generation Sequencing (NGS) of 517 kinase genes was performed using Agilent Kinome capture and run on the Illumina MiSeq at PE150bp. The NanoString nCounter® platform analyzed the expression of 519 kinase genes. Kinase activity of tissue lysates was measured using PamStation®12 high-content phospho-peptide substrate microarray system. Network mapping was done with GeneGo MetaCore™ and upstream kinase prediction was performed with BioNavigator and the Kinexus database. Ingenuity pathway analysis was performed to integrate elevated kinase activity and gene over-expression with coexisting missense mutations at DNA level. Top pathways upregulated in both the kinase activity and gene expression platforms were PTEN, STAT3, GNRH, IL-8 and B cell receptor signaling. Potentially relevant missense mutations were seen in 176 kinase genes, with the top altered pathways overlapping with gene overexpression being GNRH, NF-kB and STAT3 signaling. ERBB2, ERBB3 and SYK were altered on NGS and also exhibited elevated kinase activity. To summarize, multiplatform comprehensive analysis of kinases discovered potential drivers of PSCC and actionable therapeutic targets. Translational studies are necessary to validate the functional relevance of our data to make advances in this rare malignancy.

Host responses to tumor cells include tumor suppressing or promoting mechanisms. We sought to detail the effect of Hedgehog (Hh) pathway inhibition on the composition of the mammary tumor immune portfolio. We hypothesized that Hh signaling mediates a crosstalk between breast cancer cells and macrophages that dictates alternative polarization of macrophages and consequently supports a tumor-promoting microenvironment. We used an immunocompetent, syngeneic mouse mammary cancer model to inhibit Hh signaling with the pharmacological inhibitor, Vismodegib. Using molecular and functional assays, we identified that Hedgehog (Hh) signaling mediates a molecular crosstalk between mammary cancer cells and macrophages that culminates in alternative polarization of macrophages. We carried out an unbiased kinomics and genomics assessment to unravel changes in global kinomic and gene signatures impacted by Hh signaling. Our investigations reveal that in an immunocompetent mammary cancer model, the administration of Vismodegib led to changes in the portfolio of tumor-infiltrating immune cells. This was characterized by a marked reduction in immune-suppressive innate and adaptive cells concomitant with an enrichment of cytotoxic immune cells. Breast cancer cells induce M2 polarization of macrophages via a crosstalk mediated by Hh ligands that alters critical kinomic and genomic signatures. Macrophage depletion improved the benefit of Hedgehog inhibition on eliciting an immunogenic, pro-inflammatory profile. We define a novel role for Hh signaling in disabling anti-tumor immunity. Inhibition of Hh signaling presents with dual advantages of tumor cell-targeting as well as re-educating a dysfunctional tumor microenvironment.

Simple Summary This work establishes that ovarian tumors contain persistent oxidative DNA damage levels that can be measured using Repair Assisted Damage Detection (RADD). The persistent DNA damage correlates with increased protein expression of PD-L1, establishing a link between genomic instability and PD-L1 expression across ovarian tumors. DNA damage may be a potential biomarker for immunotherapy. Abstract Programmed death ligand-1 (PD-L1) inhibitors are currently under investigation as a potential treatment option for ovarian cancer. Although this therapy has shown promise, its efficacy is highly variable among patients. Evidence suggests that genomic instability influences the expression of PD-L1, but little is known about this relationship in ovarian cancer. To examine the relationship between PD-L1 expression and genomic instability, we measured DNA damage using Repair Assisted Damage Detection (RADD). We then correlated the presence of persistent DNA damage in the ovarian tumor with protein expression of PD-L1 using immunohistochemistry. Ovarian tumors showed a high prevalence of oxidative DNA damage. As the level of oxidative DNA damage increased, we saw a significant correlation with PD-L1 expression. The highest correlation between DNA damage and PD-L1 expression was observed for mucinous ovarian tumors (r = 0.82), but a strong correlation was also observed for high grade serous and endometrioid tumors (r = 0.67 and 0.69, respectively). These findings link genomic instability to PD-L1 protein expression in ovarian cancer and suggest that persistent DNA damage can be used as a potential biomarker for patient selection for immunotherapy treatment.

// Zheng Ping 1 , Gene P. Siegal 1, 6 , Shuko Harada 1, 6 , Isam-Eldin Eltoum 1, 6 , Mariam Youssef 1 , Tiansheng Shen 1 , Jianbo He 3 , Yingjie Huang 7 , Dongquan Chen 4, 6 , Yiping Li 2, 6 , Kirby I Bland 5, 6 , Helena R Chang 8 , Dejun Shen 1, 6 1 Division of Anatomic Pathology, University of Alabama at Birmingham, Birmingham, AL, USA 2 Division of Cell and Molecular Pathology, Departments of Pathology, University of Alabama at Birmingham, Birmingham, AL, USA 3 Division of Hematology/Oncology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA 4 Division of Preventive Medicine, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA 5 Division of Surgical Oncology, Department of Surgery, University of Alabama at Birmingham, Birmingham, AL, USA 6 UAB Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL, USA 7 Ningbo Clinical and Pathological Diagnosis Center, Ningbo, Zhejiang, China 8 Revlon/UCLA Breast Center, Department of Surgery, University of California at Los Angeles, David Geffen School of Medicine, Los Angeles, CA, USA Correspondence to: Dejun Shen, email: dshen@uabmc.edu Keywords: invasive lobular carcinoma, CDH1 mutation, ERBB2 mutation, genomics, TCGA Received: September 09, 2016 Accepted: October 24, 2016 Published: November 02, 2016 ABSTRACT E-cadherin ( CDH1 ) is a glycoprotein that mediates adhesion between epithelial cells and also suppresses cancer invasion. Mutation or deletion of the CDH1 gene has been reported in 30–60% cases of invasive lobular carcinoma (ILC). However, little is known about genomic differences between ILC with and without a CDH1 alteration. Therefore, we analyzed whole genome sequencing data of 169 ILC cases from The Cancer Genome Atlas (TCGA) to address this deficiency. Our study shows that CDH1 gene was altered in 59.2% (100/169) of ILC. No significant difference was identified between CDH1 -altered and -unaltered ILC cases for any of the examined demographic, clinical or pathologic characteristics, including histologic grade, tumor stage, lymph node metastases, or ER/PR/HER2 states. Seven recurrent mutations ( PTEN , MUC16 , ERBB2 , FAT4 , PCDHGA2 , HERC1 and FLNC ) and four chromosomal changes with recurrent copy number variation (CNV) (11q13, 17q12-21, 8p11 and 8q11) were found in ILC, which correlated with a positive or negative CDH1 alteration status, respectively. The prevalence of the most common breast cancer driver abnormalities including TP53 and PIK3CA mutations and MYC and ERBB2 amplifications showed no difference between the two groups. However, CDH1 -altered ILC with an ERBB2 mutation shows a significantly worse prognosis compared to its counterparts without such a mutation. Our study suggests that CDH1 -altered ILC patients with ERBB2 mutations may represent an actionable group of patients who could benefit from targeted breast cancer therapy.

Long-term self-renewing hematopoietic stem cell (LT-HSC) homeostasis within the bone marrow (BM) of adult mammals is regulated by complex interactions between LT-HSC and a number of niche-associated cell types including mesenchymal stromal/stem cells (MSC), osteoblasts (OB), macrophage, and neuronal cells in close proximity with the vasculature. Here, we cloned and functionally characterized a murine BM MSC subpopulation that was uniformly Nestin+Lepr +Sca-1+CD146+ and could be stably propagated with high colony-forming unit fibroblast re-cloning efficiency. MSC synergized with SCF and IL-11 to support a 20-fold expansion in true LT-HSC after 10-days of in vitro coculture. Optimal stimulation of LT-HSC expansion was minimally dependent on Notch signaling but was significantly enhanced by global inhibition of Wnt signaling. The self-renewal-promoting activity of MSC was progressively lost when MSC clones were differentiated into mature OB. This suggests that the stage of osteoblast development may significantly impact the ability of osteolineage cells to support LT-HSC homeostasis in vivo.

Purpose: In prostate cancer cells, there is CD24-dependent inactivation of mutant p53, but the mechanism and its significance remain largely unknown. Here, we validated this observation and explored the therapeutic potential of targeting CD24 in TP53 mutant prostate cancer cells. Experimental Design: Overall, 553 prostate cancers (522 formalin-fixed paraffin-embedded and 31 frozen tissues) were assessed for protein or mRNA expression of CD24 and TP53. The effects of CD24 on p53-dependent transcriptional regulation, cancer cell growth, the cell cycle, apoptosis, and mutant p53 restoration were also determined. Results: As determined with three sample cohorts, CD24 and p53 were not expressed in prostate epithelial cells but in prostate cancer cells in 48% of cases for CD24 and 16% of cases for p53 (mutant form). Expressions of CD24 and mutant p53 were more frequently observed in late-stage and metastatic prostate tumors. Mutant p53 accompanied with CD24 was expressed in most cases (91.6%, 76/83). Silencing of CD24 increased the transcriptional activity of p53 target genes, such as CDKNA1, VDR, and TP53INP1, leading to suppression of p53-dependent cell growth, cell-cycle arrest, and apoptosis in most TP53-mutant prostate cancer cells. Silencing of CD24 enhanced restoration of PRIMA-1–induced mutant p53 in endogenous TP53P223L/V274F DU145 cells and in PC3 cells transfected with TP53R273H. Conclusions: In human prostate cancers, there is CD24-dependent inactivation of mutant p53. The coexpression of CD24 and p53 may help identify aggressive cancers. Targeting CD24 provides a strategy to enhance mutant p53-restoring therapies, especially in patients with TP53R273H prostate cancer. Clin Cancer Res; 22(10); 2545–54. ©2015 AACR.

Inter-ethnic differential gene expression in stage II recurrent colorectal cancers Prachi Bajpai1, Amr Elholy1, Michael Behring1, Dongquan Chen2, 3, Kevin Hale1, Sumit Agrawal1, Hyung-Gyoon Kim1, Trafina Jadhav1, Temegsen Samuel4, Upender Manne1, 3 1Department of Pathology, 2Division of Preventive Medicine, Department of Medicine, University of Alabama at Birmingham (UAB), Birmingham, Alabama, USA, 3O’Neal Comprehensive Cancer Center of UAB, Birmingham; 4Department of Pathobiology, College of Veterinary Medicine, Tuskegee University, Tuskegee, Alabama, USA Background and Objective: For colorectal cancer (CRC), the second most common cause of cancer-related death in the US, there are racial/ethnic disparities in incidence and mortality. Of these patients, 25-35% with pathologic Stage II CRCs exhibit recurrence after surgery with curative intent. Relative to Caucasian (CA) patients, African American (AA) patients with CRC have a 20% higher stage-specific mortality. The present study delineates a distinct gene expression profile, specific for Stage II CRCs, based on disease recurrence and patient race/ethnicity. Methods: We obtained gene expression profiles for Stage II CRCs from 16 AA and 30 CA patients by the use of Affymetrix GeneChip microarrays. Samples from patients with recurrent disease, within 5 years post-surgery, were compared to those with non-recurrent CRCs within 5 years post-surgery. For comparison between AA with CA patients, gene expression was assessed. Results: For recurrent CRCs, there were nine genes common to AA and CA patients, suggesting a common gene signature specific for Stage II disease. For tumors, relative to their corresponding normal tissues, there was higher expression of five of these genes; the direction of association with disease recurrence was distinct with race/ethnicity. GFM2, DCAF17, and GEN1 had a positive association with recurrence in CAs and a negative association with recurrence in AAs. Additionally, for recurrent patients, TAPBP and FEZ were upregulated in AAs and downregulated in CAs (False Discovery Rate Citation Format: Prachi Bajpai, Amr Elholy, Michael Behring, Dongquan Chen, Kevin Hale, Sumit Agrawal, Hyung-Gyoon Kim, Trafina Jadhav, Temegsen Samuel, Upender Manne. Inter-ethnic differential gene expression in stage II recurrent colorectal cancers [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr C044.

Delicate balance between cellular plasticity and differentiation is critically maintained during mammary development. Disruption of this balance leads to breast cancer initiation and metastatic progression. Recent findings from our lab have revealed that N-Myc and STAT Interactor (NMI) protein is decreased in 70% of primary patient specimens with metastatic breast cancer. Mammary specific Nmi knock out mouse model revealed that conditional Nmi loss disrupts luminal differentiation in the mammary gland affecting alveologenesis and prompted the progression of tumors with aggressive metastatic characteristics. Our studies showed that Nmi is induced at the onset of pregnancy and its expression remains throughout lactation. Furthermore, prolactin stimulation and differentiation of HC11 murine mammary epithelial cells is accompanied by up-regulation of Nmi. STAT5a is one of the downstream effector of prolactin and is essential for differentiation of secretory alveolar epithelium. However, functional relationship of NMI and STAT5a was not known. Here we present our finding that NMI and STAT5a expression has a direct relationship in normal mammary development as well as in breast primary and metastatic tumor specimens. We demonstrate that loss of Nmi in vivo caused a decrease in STAT5a activity and a subsequent transcriptomic shift in mammary epithelial and breast cancer cells. Detailed examination of STAT5a mammary specific controlled genetic program in the context of transcription profiles of NMI knockout and overexpressing cell lines as well as mammary tumors revealed ISG20, interferon stimulated exonuclease gene 20, as a unique negatively regulated transcript. Here we show that expression of ISG20 is kept in check by NMI by miR17-92 cluster and that ISG20 has a positive influence on tumor progression and metastasis. Citation Format: Rajeev Sharad Samant, Heba A Alsheikh, Brandon J Metge, Dongquan Chen, Shi Wei, Lalita A Shevde. N-Myc and STAT interactor regulates STAT5a in mammary development and metastasis [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-14.

Advanced age has been associated with alterations to the microbiome within the intestinal tract as well as intestinal permeability (i.e., “leaky gut”). Prior studies suggest that intestinal permeability may contribute to increases in systemic inflammation—an aging hallmark—possibly via microorganisms entering the circulation. Yet, no studies exist describing the state of the circulating microbiome among older persons. To compare microbiota profiles in serum between healthy young (20–35 years, n = 24) and older adults (60–75 years, n = 24) as well as associations between differential microbial populations and prominent indices of age-related inflammation. Unweighted Unifrac analysis, a measure of β-diversity, revealed that microbial communities clustered differently between young and older adults. Several measures of α-diversity, including chao1 (p = 0.001), observed species (p = 0.001), and phylogenetic diversity (p = 0.002) differed between young and older adults. After correction for false discovery rate (FDR), age groups differed (all p values ≤ 0.016) in the relative abundance of the phyla Bacteroidetes, SR1, Spirochaetes, Bacteria_Other, TM7, and Tenericutes. Significant positive correlations (p values ≤ 0.017 after FDR correction) were observed between IGF1 and Bacteroidetes (ρ = 0.380), Spirochaetes (ρ = 0.528), SR1 (ρ = 0.410), and TM7 (ρ = 0.399). Significant inverse correlations were observed for IL6 with Bacteroidetes (ρ = − 0.398) and TM7 (ρ = − 0.423), as well as for TNFα with Bacteroidetes (ρ = − 0.344). Similar findings were observed at the class taxon. These data are the first to demonstrate that the richness and composition of the serum microbiome differ between young and older adults and that these factors are linked to indices of age-related inflammation.

In men with advanced penile squamous cell carcinoma receiving first-line chemotherapy, visceral metastases (VM) and Eastern Cooperative Oncology Group performance status ≥1 are poor prognostic factors for overall survival (OS). We hypothesized that tumor gene expression profiling may enhance prognostic stratification and identify potential therapeutic targets. In this retrospective study, RNA extracted from macrodissected tumors underwent profiling for the expression of 738 genes using NanoString. Univariate and multivariate analyses assessed the association of genes, VM, and performance status with OS. Tumors were available from 25 men who received first-line cisplatin-based chemotherapy. In univariate analysis, upregulated MAML2 ( p =0.004), KITLG ( p ≤0.0001), and JAK1 ( p =0.029) genes were associated with poor OS, and upregulated FANCA was associated with better OS ( p =0.024). In stepwise multivariate analyses, VM (hazard ratio=12.75, p =0.0001) and MAML2 (hazard ratio=10.411, p =0.003) were associated with poor OS. The presence of none, one, and both of these poor risk factors was associated with significantly different median OS of 18.4 mo, 7.2 mo, and 2.1 mo, respectively. Unsupervised clustering demonstrated two major molecular subtypes with trend for different survivals ( p =0.052). Validation of results is necessary. Patient summary The expression of the MAML2 gene in penile cancers from men receiving first-line cisplatin-based chemotherapy predicted overall survival independent of clinical factors.

// Zachary C. Dobbin 1,2 , Ashwini A. Katre 1 , Adam D. Steg 1 , Britt K. Erickson 1 , Monjri M. Shah 1 , Ronald D. Alvarez 1 , Michael G. Conner 3 , David Schneider 4 , Dongquan Chen 5 and Charles N. Landen 6 1 Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, University of Alabama at Birmingham 2 NIH Medical Scientist Training Program, University of Alabama at Birmingham 3 Department of Pathology, University of Alabama at Birmingham 4 Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham 5 Division of Preventative Medicine, Department of Medicine, University of Alabama at Birmingham 6 Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, The University of Virginia, Charlottesville, VA Correspondence: Charles N. Landen Jr, email: // Keywords : Ovarian Cancer, Patient-derived xenograft, cancer stem cells, chemoresistance, animal models of cancer Received : August 08, 2014 Accepted : August 18, 2014 Published : August 19, 2014 Abstract A cornerstone of preclinical cancer research has been the use of clonal cell lines. However, this resource has underperformed in its ability to effectively identify novel therapeutics and evaluate the heterogeneity in a patient’s tumor. The patient-derived xenograft (PDX) model retains the heterogeneity of patient tumors, allowing a means to not only examine efficacy of a therapy, but also basic tenets of cancer biology in response to treatment. Herein we describe the development and characterization of an ovarian-PDX model in order to study the development of chemoresistance. We demonstrate that PDX tumors are not simply composed of tumor-initiating cells, but recapitulate the original tumor’s heterogeneity, oncogene expression profiles, and clinical response to chemotherapy. Combined carboplatin/paclitaxel treatment of PDX tumors enriches the cancer stem cell populations, but persistent tumors are not entirely composed of these populations. RNA-Seq analysis of six pair of treated PDX tumors compared to untreated tumors demonstrates a consistently contrasting genetic profile after therapy, suggesting similar, but few, pathways are mediating chemoresistance. Pathways and genes identified by this methodology represent novel approaches to targeting the chemoresistant population in ovarian cancer

Humans are exposed to an array of chemicals via the food, drink and air, including a significant number that can mimic endogenous hormones. One such chemical is Bisphenol A (BPA), a synthetic chemical that has been shown to cause developmental alterations and to predispose for mammary cancer in rodent models. In contrast, the phytochemical genistein has been reported to suppress chemically induced mammary cancer in rodents, and Asians ingesting a diet high in soy containing genistein have lower incidence of breast and prostate cancers. In this study, we sought to: (1) identify protein biomarkers of susceptibility from blood sera of rats exposed prepubertally to BPA or genistein using Isobaric Tandem Mass Tags quantitative mass spectrometry (TMT-MS) combined with MudPIT technology and, (2) explore the relevance of these proteins to carcinogenesis. Prepubertal exposures to BPA and genistein resulted in altered expression of 63 and 28 proteins in rat sera at postnatal day (PND) 21, and of 9 and 18 proteins in sera at PND35, respectively. This study demonstrates the value of using quantitative proteomic techniques to explore the effect of chemical exposure on the rat serum proteome and its potential for unraveling cellular targets altered by BPA and genistein involved in carcinogenesis.

SUMMARY Unresectable glioblastoma (GBM) cells in the invading tumor edge can act as seeds for recurrence. The molecular and phenotypic properties of these cells remain elusive. Here, we report that the invading edge and tumor core have two distinct types of glioma stem-like cells (GSCs) that resemble proneural (PN) and mesenchymal (MES) subtypes, respectively. Upon exposure to ionizing radiation (IR), GSCs, initially enriched for a CD133+ PN signature, transition to a CD109+ MES subtype in a C/EBP-β-dependent manner. Our gene expression analysis of paired cohorts of patients with primary and recurrent GBMs identified a CD133-to-CD109 shift in tumors with an MES recurrence. Patient-derived CD133−/CD109+ cells are highly enriched with clonogenic, tumor-initiating, and radiation-resistant properties, and silencing CD109 significantly inhibits these phenotypes. We also report a conserved regulation of YAP/TAZ pathways by CD109 that could be a therapeutic target in GBM., Graphical Abstract, In Brief Minata et al., in response to the proinflammatory environment induced by radiation, find that the tumor cells at the invasive edge acquire the expression of the CD109 protein concomitantly losing CD133. CD109 drives oncogenic signaling through the YAP/TAZ pathway, confers radioresistance to the cells, and represents a new potential therapeutic target for glioblastoma.

TLR9 is a cellular DNA-receptor, which is widely expressed in breast and other cancers. Although synthetic TLR9-ligands induce cancer cell invasion in vitro, the role of TLR9 in cancer pathophysiology has remained unclear. We show here that living cancer cells uptake DNA from chemotherapy-killed cancer cells. We discovered that such DNA induces TLR9- and cathepsin-mediated invasion in living cancer cells. To study whether this phenomenon contributes to treatment responses, triple-negative, human MDA-MB-231 breast cancer cells stably expressing control, or TLR9 siRNA were inoculated orthotopically into nude mice. The mice were treated with vehicle or doxorubicin. The tumor groups exhibited equal decreases in size in response to doxorubicin. However, while the weights of vehicle-treated mice were similar, mice bearing control siRNA tumors became significantly more cachectic in response to doxorubicin, as compared with similarly treated mice bearing TLR9 siRNA tumors, suggesting a TLR9-mediated inflammation at the site of the tumor. In conclusion, our findings propose that DNA released from chemotherapy-killed cancer cells has significant influence on TLR9-mediated biological effects in living cancer cells. Through these mechanisms, tumor TLR9 expression may affect treatment responses to chemotherapy.

Accumulating evidence suggests that glioma stem cells (GSCs) are important therapeutic targets in glioblastoma (GBM). In this study, we identified NIMA-related kinase 2 (NEK2) as a functional binding protein of enhancer of zeste homolog 2 (EZH2) that plays a critical role in the posttranslational regulation of EZH2 protein in GSCs. NEK2 was among the most differentially expressed kinase-encoding genes in GSC-containing cultures (glioma spheres), and it was required for in vitro clonogenicity, in vivo tumor propagation, and radioresistance. Mechanistically, the formation of a protein complex comprising NEK2 and EZH2 in glioma spheres phosphorylated and then protected EZH2 from ubiquitination-dependent protein degradation in a NEK2 kinase activity–dependent manner. Clinically, NEK2 expression in patients with glioma was closely associated with EZH2 expression and correlated with a poor prognosis. NEK2 expression was also substantially elevated in recurrent tumors after therapeutic failure compared with primary untreated tumors in matched GBM patients. We designed a NEK2 kinase inhibitor, compound 3a (CMP3a), which efficiently attenuated GBM growth in a mouse model and exhibited a synergistic effect with radiotherapy. These data demonstrate a key role for NEK2 in maintaining GSCs in GBM by stabilizing the EZH2 protein and introduce the small-molecule inhibitor CMP3a as a potential therapeutic agent for GBM.

Histologic grade is one of the most important microscopic features used to predict the prognosis of invasive breast cancer and may serve as a marker for studying cancer driving genomic abnormalities in vivo. We analyzed whole genome sequencing data from 680 cases of TCGA invasive ductal carcinomas of the breast and correlated them to corresponding pathology information. Ten genetic abnormalities were found to be statistically associated with histologic grade, including three most prevalent cancer driver events, TP53 and PIK3CA mutations and MYC amplification. A distinct genetic interaction among these genomic abnormalities was revealed as measured by the histologic grading score. While TP53 mutation and MYC amplification were synergistic in promoting tumor progression, PIK3CA mutation was found to have alleviated the oncogenic effect of either the TP53 mutation or MYC amplification, and was associated with a significant reduction in mitotic activity in TP53 mutated and/or MYC amplified breast cancer. Furthermore, we discovered that different types of genetic abnormalities (mutation versus amplification) within the same cancer driver gene (PIK3CA or GATA3) were associated with opposite histologic changes in invasive breast cancer. In conclusion, our study suggests that histologic grade may serve as a biomarker to define cancer driving genetic events in vivo.

Kinases are therapeutically actionable targets. Kinase inhibitors targeting vascular endothelial growth factor receptors (VEGFR) and mammalian target of rapamycin (mTOR) improve outcomes in metastatic clear cell renal cell carcinoma (ccRCC), but are not curative. Metastatic tumor tissue has not been comprehensively studied for kinase gene expression. Paired intra-patient kinase gene expression analysis in primary tumor (T), matched normal kidney (N) and metastatic tumor tissue (M) may assist in identifying drivers of metastasis and prioritizing therapeutic targets. We compared the expression of 519 kinase genes using NanoString in T, N and M in 35 patients to discover genes over-expressed in M compared to T and N tissue. RNA-seq data derived from ccRCC tumors in The Cancer Genome Atlas (TCGA) were used to demonstrate differential expression of genes in primary tumor tissue from patients that had metastasis at baseline (n = 79) compared to those that did not develop metastasis for at least 2 years (n = 187). Functional analysis was conducted to identify key signaling pathways by using Ingenuity Pathway Analysis. Of 10 kinase genes overexpressed in metastases compared to primary tumor in the discovery cohort, 9 genes were also differentially expressed in TCGA primary tumors with metastasis at baseline compared to primary tumors without metastasis for at least 2 years: EPHB2, AURKA, GSG2, IKBKE, MELK, CSK, CHEK2, CDC7 and MAP3K8; p

Toll-like receptor-9 (TLR9) is a DNA receptor widely expressed in cancers. Although synthetic TLR9 ligands induce cancer cell invasion in vitro, the role of TLR9 in cancer pathophysiology is unclear. We discovered that low tumor TLR9 expression is associated with significantly shortened disease-specific survival in patients with triple negative but not with ER+ breast cancers. A likely mechanism of this clinical finding involves differential responses to hypoxia. Our pre-clinical studies indicate that while TLR9 expression is hypoxia-regulated, low TLR9 expression has different effects on triple negative and ER+ breast cancer invasion in hypoxia. Hypoxia-induced invasion is augmented by TLR9 siRNA in triple negative, but not in ER+ breast cancer cells. This is possibly due to differential TLR9-regulated TIMP-3 expression, which remains detectable in ER+ cells but disappears from triple-negative TLR9 siRNA cells in hypoxia. Our results demonstrate a novel role for this innate immunity receptor in cancer biology and suggest that TLR9 expression may be a novel marker for triple-negative breast cancer patients who are at a high risk of relapse. Furthermore, these results suggest that interventions or events, which induce hypoxia or down-regulate TLR9 expression in triple-negative breast cancer cells may actually induce their spread.

In experimental autoimmune encephalomyelitis (EAE) and other neurodegenerative diseases, astrocytes play an important role in promoting or attenuating the inflammatory response through induction of different cytokines and growth factors. HuR plays a major role in regulating many of these factors by modulating RNA stability and translational efficiency. Here, we engineered transgenic mice to express HuR in astrocytes using the human glial fibrillary acidic protein promoter and found that female transgenic mice had significantly less clinical disability and histopathological changes in the spinal cord. Ovariectomy prior to EAE induction abrogated the protective effect. Our findings support a role for the astrocyte and posttranscriptional regulation in hormonally-mediated attenuation of EAE.

Precis: Many genes are differentially expressed in normal compared to anencephalic human fetal adrenals (HFAs), especially the Fras-1-related extracellular matrix protein (FREM2) gene. FREM2 expression appears to be regulated by adrenocorticotrophic hormone (ACTH). Context: The expression profiles of genes responsible for cortical growth and zonation in the HFA gland are poorly characterized. The neural tube disorder anencephaly is associated with fetal adrenal hypoplasia with a large size reduction of the fetal zone of the HFA. Objective: To determine gene expression profile differences in the adrenals of anencephalic compared to normal HFAs to identify genes that may play important roles in adrenal development. Design and Methods: Fresh tissues were obtained at the time of autopsy from normal and anencephalic human fetuses delivered at mid-gestation. The following techniques were used: cell culture, messenger RNA (mRNA) extraction, microarray analysis, complementary DNA (cDNA) synthesis, quantitative real-time reverse transcriptase polymerase chain reaction (QT-PCR). Results: We identified over 40 genes expressed at levels 4-fold or greater in the normal versus anencephalic HFAs and that 28 genes were expressed at increased levels in the anencephalic HFA. The expression of FREM2 at approximately 40-fold greater levels in the normal HFA compared to the HFA of anencephalic fetuses was confirmed by QT-PCR. Expression of FREM2 in the kidney was not significantly different between normal and anencephalic fetuses. In cultured HFA cells, ACTH treatment for 48 hours increased the expression of FREM2 and a gene responsive to ACTH, CYP17, but not tyrosine hydroxylase. Conclusions: Abnormal expression of many genes may be involved in the adrenal hypoplasia seen in anencephaly. FREM2 appears to be regulated by ACTH and is the most differentially expressed gene, which may be important in the development and function of the HFA, particularly the fetal zone of the HFA.

Cardiac dysfunction and mortality associated with trauma and sepsis increase with age. Mitochondria play a critical role in the energy demand of cardiac muscles, and thereby on the function of the heart. Specific molecular pathways responsible for mitochondrial functional alterations after injury in relation to aging are largely unknown. To further investigate this, 6- and 22-month-old rats were subjected to trauma-hemorrhage (T-H) or sham operation and euthanized following resuscitation. Left ventricular tissue was profiled using our custom rodent mitochondrial gene chip (RoMitochip). Our experiments demonstrated a declined left ventricular performance and decreased alteration in mitochondrial gene expression with age following T-H and we have identified c-Myc, a pleotropic transcription factor, to be the most upregulated gene in 6- and 22-month-old rats after T-H. Following T-H, while 142 probe sets were altered significantly (39 up and 103 down) in 6-month-old rats, only 66 were altered (30 up and 36 down) in 22-month-old rats; 36 probe sets (11 up and 25 down) showed the same trend in both groups. The expression of c-Myc and cardiac death promoting gene Bnip3 were increased, and Pgc1-α and Ppar-α a decreased following T-H. Eleven tRNA transcripts on mtDNA were upregulated following T-H in the aged animals, compared with the sham group. Our observations suggest a c-myc–regulated mitochondrial dysfunction following T-H injury and marked decrease in age-dependent changes in the transcriptional profile of mitochondrial genes following T-H, possibly indicating cellular senescence. To our knowledge, this is the first report on mitochondrial gene expression profile following T-H in relation to aging.

Triple-negative breast cancer (TNBC) is an aggressive cancer with poor prognosis and is difficult to treat. There is a major health disparity associated with TNBC; the number of fatalities associated with this disease is significantly higher in African American women compared to their Caucasian counterparts. The mechanisms behind these differences have not been entirely elucidated. There are no targeted therapies for TNBC; instead, standard therapy for this disease includes a combination of chemotherapeutic drugs: doxorubicin (DOX), paclitaxel (TAX), and/or cyclophosphamide (CTX). Even more troubling, the efficacy of standard TNBC therapies is marginal and patients eventually become resistant to this intervention. TNBC patients suffer from chemoresistance and metastasis and ultimately succumb to this disease. Understanding TNBC and its health disparity requires a better understanding of the race-specific differences in breast tumor biology, molecules leading to breast inflammation, and the mechanisms associated with TNBC chemoresistance. Chemokines and their receptors play a significant role in TNBC. Our laboratory was the first to show that CXCL13-CXCR5 signaling mediates prostate cancer cell growth, migration, invasion, and survival (1-3). We also demonstrated that CXCL13-CXCR5 signaling induces cancer progression signaling pathways PI3K, AKT, ERK, and Jun (1;4). These preliminary and published findings provide the rationale or scientific premise in support of our hypothesis that CXCL13-CXCR5 signaling promotes TNBC progression migration, invasion, proliferation, and chemoresistance. To determine whether CXCL13-CXCR5 signaling induces TNBC, the expression profile of CXCL13 and CXCR5 RNA, associated miRNAs, and cancer progression genes (epithelial mesenchymal transition [EMT] markers) will be correlated with disease-free and overall survival of TNBC patients in silico using RNA-seq datasets from the Sequence Read Archive (SRA). Fastq data set files were downloaded from SRA, checked for quality control, aligned, and normalized using open-source platforms. Hierarchical clustering, gene set enrichment analysis, disease progression analysis, and statistics were conducted using Partek Genomic Suite. Pathway enrichment analysis was conducted using Ingenuity Pathway Studios. Overall, CXCL13-CXCR5 signaling is implicated in TNBC progression, migration, and invasion. Citation Format: Courtney D. Dill, Adaugo Okoro, Dongquan Chen, James W. Lillard, Jr.. The role of CXCL13-CXCR5 signaling in triple-negative breast cancer progression [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr B50.

Herein, we report our examination of the anti-breast cancer activity of a novel synthetic compound, 7-(benzylamino)-1, 3, 4, 8-tetrahydropyrrolo [4, 3, 2-de]quinolin-8(1H)-one (BA-TPQ). This agent is an analog of a naturally occurring marine compound, and was found to be the most active out of more than 40 related compounds. We investigated the in vitro activity of BA-TPQ on the survival, proliferation, and apoptosis of breast cancer cells using the MTT and BrdUrd assays, and Annexin/Annexin-PI staining and flow cytometry. The in vivo anti-cancer effects of BA-TPQ were evaluated in xenograft models of breast cancer. Finally, the mechanisms of action of the compound were also assessed by cDNA microarrays, RT-PCR and Western blotting. In a dose-dependent manner, BA-TPQ inhibited cell growth and induced apoptosis and cell cycle arrest in human MCF-7 and MDA-MB-468 breast cancer cells in vitro, and showed in vivo efficacy in mice bearing MCF-7 or MDA-MB-468 xenograft tumors. We demonstrated that BA-TPQ modifies the expression of numerous molecules involved in cell cycle progression and apoptosis. Similar changes in protein expression were observed in vitro and in vivo, as determined by examination of cells and excised xenograft tumors. Our preclinical data indicate that BA-TPQ is a potential therapeutic agent for breast cancer that has multiple hormone-, Her2-, and p53-independent mechanisms of action, providing a basis for further development of the compound as a novel anticancer agent.

BACKGROUND Although anti-angiogenic therapy is a promising new line of therapy for prostate cancer, we recently reported that stable expression of endostatin arrested the progression of prostate cancer to poorly differentiated state and distant metastasis in TRAMP mice. However, the same therapy failed to provide any benefit when given either during or after the onset of metastatic switch. The present study determined the possible mechanisms behind the selective advantage of endostatin therapy in early-stage disease. METHODS Angiogenesis-related gene expression analysis was performed to identify target genes and molecular pathways involved in the therapy effects. Based on the results from in vivo studies, and recapitulation of the in vivo data in vitro using tumorigenic and non-tumorigenic human prostate cancer cells that are either androgen-sensitive or androgen-independent, analyses of possible mechanisms of the selective advantage of early treatment were performed using assays for cell proliferation, apoptosis, migration, and cell signaling. The identified mechanisms were further confirmed in vivo. RESULTS Results indicated that cells with high androgen receptor (AR) expression were more sensitive to endostatin treatment than androgen-independent cells with low or no AR expression. Endostatin was found to significantly downregulate the expression of growth factors, receptor tyrosine kinases, proteases, and AR both in vitro and in vivo only when the cells express high-levels of AR. Cell proliferation was not influenced by endostatin treatment but migration was significantly affected only in androgen-sensitive cells. Targeted downregulation of AR prior to endostatin treatment in androgen-sensitive cells and overexpression of AR in androgen-independent cells indicated that the effect of endostatin via AR downregulation is mediated by a non-genotropic mechanism on Ras and RhoA pathways, and independently of AR on MAPK/ERK pathway. CONCLUSIONS These data indicate that systemically stable endostatin expression delays the onset of metastatic switch by acting on multiple pathways involving AR. Prostate 69:1055–1066, 2009. © 2009 Wiley-Liss, Inc.