Multi-environment Genomic Selection in Rice Elite Breeding Lines.

BACKGROUND: Assessing the performance of elite lines in target environments is essential for breeding programs to select the most relevant genotypes. One of the main complexities in this task resides in accounting for the genotype by environment interactions. Genomic prediction models that integrate information from multi-environment trials and environmental covariates can be efficient tools in this context. The objective of this study was to assess the predictive ability of different genomic prediction models to optimize the use of multi-environment information. We used 111 elite breeding lines representing the diversity of the international rice research institute breeding program for irrigated ecosystems. The lines were evaluated for three traits (days to flowering, plant height, and grain yield) in 15 environments in Asia and Africa and genotyped with 882 SNP markers. We evaluated the efficiency of genomic prediction to predict untested environments using seven multi-environment models and three cross-validation scenarios. RESULTS: The elite lines were found to belong to the indica group and more specifically the indica-1B subgroup which gathered improved material originating from the Green Revolution. Phenotypic correlations between environments were high for days to flowering and plant height (33% and 54% of pairwise correlation greater than 0.5) but low for grain yield (lower than 0.2 in most cases). Clustering analyses based on environmental covariates separated Asia's and Africa's environments into different clusters or subclusters. The predictive abilities ranged from 0.06 to 0.79 for days to flowering, 0.25-0.88 for plant height, and - 0.29-0.62 for grain yield. We found that models integrating genotype-by-environment interaction effects did not perform significantly better than models integrating only main effects (genotypes and environment or environmental covariates). The different cross-validation scenarios showed that, in most cases, the use of all available environments gave better results than a subset. CONCLUSION: Multi-environment genomic prediction models with main effects were sufficient for accurate phenotypic prediction of elite lines in targeted environments. These results will help refine the testing strategy to update the genomic prediction models to improve predictive ability.

Identification of an Elite Core Panel as a Key Breeding Resource to Accelerate the Rate of Genetic Improvement for Irrigated Rice.

Rice genetic improvement is a key component of achieving and maintaining food security in Asia and Africa in the face of growing populations and climate change. In this effort, the International Rice Research Institute (IRRI) continues to play a critical role in creating and disseminating rice varieties with higher productivity. Due to increasing demand for rice, especially in Africa, there is a strong need to accelerate the rate of genetic improvement for grain yield. In an effort to identify and characterize the elite breeding pool of IRRI's irrigated rice breeding program, we analyzed 102 historical yield trials conducted in the Philippines during the period 2012-2016 and representing 15,286 breeding lines (including released varieties). A mixed model approach based on the pedigree relationship matrix was used to estimate breeding values for grain yield, which ranged from 2.12 to 6.27 t·ha-1. The rate of genetic gain for grain yield was estimated at 8.75 kg·ha-1 year-1 (0.23%) for crosses made in the period from 1964 to 2014. Reducing the data to only IRRI released varieties, the rate doubled to 17.36 kg·ha-1 year-1 (0.46%). Regressed against breeding cycle the rate of gain for grain yield was 185 kg·ha-1 cycle-1 (4.95%). We selected 72 top performing lines based on breeding values for grain yield to create an elite core panel (ECP) representing the genetic diversity in the breeding program with the highest heritable yield values from which new products can be derived. The ECP closely aligns with the indica 1B sub-group of Oryza sativa that includes most modern varieties for irrigated systems. Agronomic performance of the ECP under multiple environments in Asia and Africa confirmed its high yield potential. We found that the rate of genetic gain for grain yield found in this study was limited primarily by long cycle times and the direct introduction of non-improved material into the elite pool. Consequently, the current breeding scheme for irrigated rice at IRRI is based on rapid recurrent selection among highly elite lines. In this context, the ECP constitutes an important resource for IRRI and NAREs breeders to carefully characterize and manage that elite diversity.

Cyclin D1 integrates G9a-mediated histone methylation.

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.

Dachshund Depletion Disrupts Mammary Gland Development and Diverts the Composition of the Mammary Gland Progenitor Pool.

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 ß (TGF-ß) 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-ß activation, was reduced by endogenous DACH1. DACH1 increases basal cells, enhances ductal formation and restrains TGF-ß activity in vivo.

Stromal cyclin D1 promotes heterotypic immune signaling and breast cancer growth.

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.

Caveolin-1 regulates the anti-atherogenic properties of macrophages.

Atherosclerosis is a complex disease initiated by the vascular accumulation of lipoproteins in the sub-endothelial space, followed by the infiltration of monocytes into the arterial intima. Caveolin-1 (Cav-1) plays an essential role in the regulation of cellular cholesterol metabolism and of various signaling pathways. In order to study specifically the role of macrophage Cav-1 in atherosclerosis, we used Cav-1 (-/-) Apoe (-/-) mice and transplanted them with bone marrow (BM) cells obtained from Cav-1 (+/+) Apoe (-/-) or Cav-1 (-/-) Apoe (-/-) mice and vice versa. We found that Cav-1 (+/+) mice harboring Cav-1 (-/-) BM-derived macrophages developed significantly larger lesions than Cav-1 (+/+) mice harboring Cav-1 (+/+) BM-derived macrophages. Cav-1 (-/-) macrophages were more susceptible to apoptosis and more prone to induce inflammation. The present study provides clear evidence that the absence of Cav-1 in macrophage is pro-atherogenic, whereas its absence in endothelial cells protects against atherosclerotic lesion formation. These findings demonstrate the cell-specific role of Cav-1 during the development of this disease.

Identification of a cyclin D1 network in prostate cancer that antagonizes epithelial-mesenchymal restraint.

Improved clinical management of prostate cancer has been impeded by an inadequate understanding of molecular genetic elements governing tumor progression. Gene signatures have provided improved prognostic indicators of human prostate cancer. The TGF-ß/BMP-SMAD4 signaling pathway, which induces epithelial-mesenchymal transition (EMT), is known to constrain prostate cancer progression induced by Pten deletion. Herein, cyclin D1 inactivation reduced cellular proliferation in the murine prostate in vivo and in isogenic oncogene-transformed prostate cancer cell lines. The in vivo cyclin D1-mediated molecular signature predicted poor outcome of recurrence-free survival for patients with prostate cancer (K-means HR, 3.75, P = 0.02) and demonstrated that endogenous cyclin D1 restrains TGF-ß, Snail, Twist, and Goosecoid signaling. Endogenous cyclin D1 enhanced Wnt and ES cell gene expression and expanded a prostate stem cell population. In chromatin immunoprecipitation sequencing, cyclin D1 occupied genes governing stem cell expansion and induced their transcription. The coordination of EMT restraining and stem cell expanding gene expression by cyclin D1 in the prostate may contribute to its strong prognostic value for poor outcome in biochemical-free recurrence in human prostate cancer.

Cav1 suppresses tumor growth and metastasis in a murine model of cutaneous SCC through modulation of MAPK/AP-1 activation.

Caveolin-1 (Cav1) is a scaffolding protein that serves to regulate the activity of several signaling molecules. Its loss has been implicated in the pathogenesis of several types of cancer, but its role in the development and progression of cutaneous squamous cell carcinoma (cSCC) remains largely unexplored. Herein, we use the keratinocyte cell line PAM212, a murine model of cSCC, to determine the function of Cav1 in skin tumor biology. We first show that Cav1 overexpression decreases cell and tumor growth, whereas Cav1 knockdown increases these attributes in PAM212 cells. In addition, Cav1 knockdown increases the invasive ability and incidence of spontaneous lymph node metastasis. Finally, we demonstrate that Cav1 knockdown increases extracellular signaling-related kinase 1/2 mitogen-activated protein kinase/activator protein-1 pathway activation. We attribute the growth and invasive advantage conferred by Cav1 knockdown to increased expression of activator protein-1 transcriptional targets, including cyclin D1 and keratin 18, which show inverse expression in PAM212 based on the expression level of Cav1. In summary, we demonstrate that loss of Cav1 affects several characteristics associated with aggressive human skin tumors and that this protein may be an important modulator of tumor growth and invasion in cSCC.

Genetic ablation of Cav1 differentially affects melanoma tumor growth and metastasis in mice: role of Cav1 in Shh heterotypic signaling and transendothelial migration.

Both cell-autonomous and non-cell-autonomous factors contribute to tumor growth and metastasis of melanoma. The function of caveolin-1 (Cav1), a multifunctional scaffold protein known to modulate several biologic processes in both normal tissue and cancer, has been recently investigated in melanoma cancer cells, but its role in the melanoma microenvironment remains largely unexplored. Here, we show that orthotopic implantation of B16F10 melanoma cells in the skin of Cav1KO mice increases tumor growth, and co-injection of Cav1-deficient dermal fibroblasts with melanoma cells is sufficient to recapitulate the tumor phenotype observed in Cav1KO mice. Using indirect coculture experiments with fibroblasts and melanoma cells combined with cytokine analysis, we found that Cav1-deficient fibroblasts promoted the growth of melanoma cells via enhanced paracrine cytokine signaling. Specifically, Cav1-deficient fibroblasts displayed increased ShhN expression, which heterotypically enhanced the Shh signaling pathway in melanoma cells. In contrast to primary tumor growth, the ability of B16F10 melanoma cells to form lung metastases was significantly reduced in Cav1KO mice. This phenotype was associated mechanistically with the inability of melanoma cells to adhere to and to transmigrate through a monolayer of endothelial cells lacking Cav1. Together, our findings show that Cav1 may regulate different mechanisms during primary melanoma tumor growth and metastatic dissemination.

Genome-wide DNA polymorphisms in elite indica rice inbreds discovered by whole-genome sequencing.

Advances in next-generation sequencing technologies have aided discovery of millions of genome-wide DNA polymorphisms, single nucleotide polymorphisms (SNPs) and insertions-deletions (InDels), which are an invaluable resource for marker-assisted breeding. Whole-genome resequencing of six elite indica rice inbreds (three cytoplasmic male sterile and three restorer lines) resulted in the generation of 338 million 75-bp paired-end reads, which provided 85.4% coverage of the Nipponbare genome. A total of 2 819 086 nonredundant DNA polymorphisms including 2 495 052 SNPs, 160 478 insertions and 163 556 deletions were discovered between the inbreds and Nipponbare, providing an average of 6.8 SNPs/kb across the genome. Distribution of SNPs and InDels in the chromosome was nonrandom with SNP-rich and SNP-poor regions being evident across the genome. A contiguous 4.3-Mb region on chromosome 5 with extremely low SNP density was identified. Overall, 83 262 nonsynonymous SNPs spanning 16 379 genes and 3620 nonsynonymous InDels in 2625 genes have been discovered which provide valuable insights into the basis underlying performance of the inbreds and the hybrids between these inbred combinations. SNPs and InDels discovered from this diverse set of indica rice inbreds not only enrich SNP resources for molecular breeding but also enable the study of genome-wide variations on hybrid performance.

Mammary gland selective excision of c-jun identifies its role in mRNA splicing.

The c-jun gene regulates cellular proliferation and apoptosis via direct regulation of cellular gene expression. Alternative splicing of pre-mRNA increases the diversity of protein functions, and alternate splicing events occur in tumors. Here, by targeting the excision of the endogenous c-jun gene within the mouse mammary epithelium, we have identified its selective role as an inhibitor of RNA splicing. Microarray-based assessment of gene expression, on laser capture microdissected c-jun(-/-) mammary epithelium, showed that endogenous c-jun regulates the expression of approximately 50 genes governing RNA splicing. In addition, genome-wide splicing arrays showed that endogenous c-jun regulated the alternate exon of approximately 147 genes, and 18% of these were either alternatively spliced in human tumors or involved in apoptosis. Endogenous c-jun also was shown to reduce splicing activity, which required the c-jun dimerization domain. Together, our findings suggest that c-jun directly attenuates RNA splicing efficiency, which may be of broad biologic importance as alternative splicing plays an important role in both cancer development and therapy resistance.

c-Jun is required for TGF-ß-mediated cellular migration via nuclear Ca²âº signaling.

Tumor progression involves the acquisition of invasiveness through a basement membrane. The c-jun proto-oncogene is overexpressed in human tumors and has been identified at the leading edge of human breast tumors. TGF-ß plays a bifunctional role in tumorigenesis and cellular migration. Although c-Jun and the activator protein 1 (AP-1) complex have been implicated in human cancer, the molecular mechanisms governing cellular migration via c-Jun and the role of c-Jun in TGF-ß signaling remains poorly understood. Here, we analyze TGF-ß mediated cellular migration in mouse embryo fibroblasts using floxed c-jun transgenic mice. We compared the c-jun wild type with the c-jun knockout cells through the use of Cre recombinase. Herein, TGF-ß stimulated cellular migration and intracellular calcium release requiring endogenous c-Jun. TGF-ß mediated Ca(2+) release was independent of extracellular calcium and was suppressed by both U73122 and neomycin, pharmacological inhibitors of the breakdown of PIP(2) into IP(3). Unlike TGF-ß-mediated Ca(2+) release, which was c-Jun dependent, ATP mediated Ca(2+) release was c-Jun independent. These studies identify a novel pathway by which TGF-ß regulates cellular migration and Ca(2+) release via endogenous c-Jun.

Cell fate determination factor Dachshund reprograms breast cancer stem cell function.

The cell fate determination factor Dachshund was cloned as a dominant inhibitor of the hyperactive epidermal growth factor receptor ellipse. The expression of Dachshund is lost in human breast cancer associated with poor prognosis. Breast tumor-initiating cells (TIC) may contribute to tumor progression and therapy resistance. Here, endogenous DACH1 was reduced in breast cancer cell lines with high expression of TIC markers and in patient samples of the basal breast cancer phenotype. Re-expression of DACH1 reduced new tumor formation in serial transplantations in vivo, reduced mammosphere formation, and reduced the proportion of CD44(high)/CD24(low) breast tumor cells. Conversely, lentiviral shRNA to DACH1 increased the breast (B)TIC population. Genome-wide expression studies of mammary tumors demonstrated DACH1 repressed a molecular signature associated with stem cells (SOX2, Nanog, and KLF4) and genome-wide ChIP-seq analysis identified DACH1 binding to the promoter of the Nanog, KLF4, and Lin28 genes. KLF4/c-Myc and Oct4/Sox2 antagonized DACH1 repression of BTIC. Mechanistic studies demonstrated DACH1 directly repressed the Nanog and Sox2 promoters via a conserved domain. Endogenous DACH1 regulates BTIC in vitro and in vivo.

The canonical NF-kappaB pathway governs mammary tumorigenesis in transgenic mice and tumor stem cell expansion.

The role of mammary epithelial cell (MEC) NF-κB in tumor progression in vivo is unknown, as murine NF-κB components and kinases either are required for murine survival or interfere with normal mammary gland development. As NF-κB inhibitors block both tumor-associated macrophages (TAM) and MEC NF-κB, the importance of MEC NF-κB to tumor progression in vivo remained to be determined. Herein, an MEC-targeted inducible transgenic inhibitor of NF-κB (IκBαSR) was developed in ErbB2 mammary oncomice. Inducible suppression of NF-κB in the adult mammary epithelium delayed the onset and number of new tumors. Within similar sized breast tumors, TAM and tumor neoangiogenesis was reduced. Coculture experiments demonstrated MEC NF-κB enhanced TAM recruitment. Genome-wide expression and proteomic analysis showed that IκBαSR inhibited tumor stem cell pathways. IκBαSR inhibited breast tumor stem cell markers in transgenic tumors, reduced stem cell expansion in vitro, and repressed expression of Nanog and Sox2 in vivo and in vitro. MEC NF-κB contributes to mammary tumorigenesis. As we show that NF-κB contributes to expansion of breast tumor stem cells and heterotypic signals that enhance TAM and vasculogenesis, these processes may contribute to NF-κB-dependent mammary tumorigenesis.

C-jun inhibits mammary apoptosis in vivo.

c-jun, which is overexpressed in a number of human cancers encodes a critical component of the AP-1 complex. c-jun has been shown to either induce or inhibit cellular apoptosis. Germ line deletion of both c-jun alleles is embryonically lethal. To determine the role of the endogenous c-jun gene in apoptosis, we performed mammary epithelial cell-targeted somatic deletion using floxed c-jun (c-jun(f/f)) conditional knockout mice. Laser capture microdissection demonstrated endogenous c-jun inhibits expression of apoptosis inducing genes and reactive oxygen species (ROS)-reducing genes (MnSOD, catalase). ROS have been implicated in apoptosis and undergo enzymatic elimination via MnSOD and CuZnSOD with further detoxification via catalase. c-jun-mediated survival was in part dependent on ROS production. c-jun-mediated repression of MnSOD and catalase occurred via mitochondrial complex I and NOX I. Collectively, these studies define a pivotal role of endogenous c-jun in promoting cell survival via maintaining mitochondrial integrity and expression of the key regulators of ROS production.

microRNA 17/20 inhibits cellular invasion and tumor metastasis in breast cancer by heterotypic signaling.

microRNAs are thought to regulate tumor progression and invasion via direct interaction with target genes within cells. Here the microRNA17/20 cluster is shown to govern cellular migration and invasion of nearby cells via heterotypic secreted signals. microRNA17/20 abundance is reduced in highly invasive breast cancer cell lines and node-positive breast cancer specimens. Cell-conditioned medium from microRNA17/20-overexpressing noninvasive breast cancer cell MCF7 was sufficient to inhibit MDA-MB-231 cell migration and invasion through inhibiting secretion of a subset of cytokines, and suppressing plasminogen activation via inhibition of the secreted plasminogen activators (cytokeratin 8 and alpha-enolase). microRNA17/20 directly repressed IL-8 by targeting its 3' UTR, and inhibited cytokeratin 8 via the cell cycle control protein cyclin D1. At variance with prior studies, these results demonstrated a unique mechanism of how the altered microRNA17/20 expression regulates cellular secretion and tumor microenvironment to control migration and invasion of neighboring cells in breast cancer. These findings not only reveal an antiinvasive function of miR-17/20 in breast cancer, but also identify a heterotypic secreted signal that mediates the microRNA regulation of tumor metastasis.

Synthesis and cytotoxicity evaluation of (tetrahydro-beta-carboline)-1,3,5-triazine hybrids as anticancer agents.

A series of tetrahydro-beta-carbolines and 1,3,5-triazine hybrids have been synthesized and evaluated for their cytotoxicity against a panel of eight human cancer cell lines and normal human fibroblasts (NIH3T3). It led us to discovery of racemic compounds 69, 71 and 75, which are selectively cytotoxic towards KB (oral cancer) cell line with IC50 values of 105.8, 664.7 and 122.2 nM, respectively; while their enantiopure forms are less active and not selective. Enantiopure compound 42 showed 2.5 times more selectivity towards MCF7 cells over normal fibroblast NIH3T3 cells with an IC50 value of 740 nM, also arrests cell cycle in G1 phase and induces apoptosis in MCF7 and MDA MB231 cell lines.

c-Jun induces mammary epithelial cellular invasion and breast cancer stem cell expansion.

The molecular mechanisms governing breast tumor cellular self-renewal contribute to breast cancer progression and therapeutic resistance. The ErbB2 oncogene is overexpressed in approximately 30% of human breast cancers. c-Jun, the first cellular proto-oncogene, is overexpressed in human breast cancer. However, the role of endogenous c-Jun in mammary tumor progression is unknown. Herein, transgenic mice expressing the mammary gland-targeted ErbB2 oncogene were crossed with c-jun(f/f) transgenic mice to determine the role of endogenous c-Jun in mammary tumor invasion and stem cell function. The excision of c-jun by Cre recombinase reduced cellular migration, invasion, and mammosphere formation of ErbB2-induced mammary tumors. Proteomic analysis identified a subset of secreted proteins (stem cell factor (SCF) and CCL5) induced by ErbB2 expression that were dependent upon endogenous c-Jun expression. SCF and CCL5 were identified as transcriptionally induced by c-Jun. CCL5 rescued the c-Jun-deficient breast tumor cellular invasion phenotype. SCF rescued the c-Jun-deficient mammosphere production. Endogenous c-Jun thus contributes to ErbB2-induced mammary tumor cell invasion and self-renewal.

p21CIP1 attenuates Ras- and c-Myc-dependent breast tumor epithelial mesenchymal transition and cancer stem cell-like gene expression in vivo.

p21(CIP1/WAF1) is a downstream effector of tumor suppressors and functions as a cyclin-dependent kinase inhibitor to block cellular proliferation. Breast tumors may derive from self-renewing tumor-initiating cells (BT-ICs), which contribute to tumor progression, recurrence, and therapy resistance. The role of p21(CIP1) in regulating features of tumor stem cells in vivo is unknown. Herein, deletion of p21(CIP1), which enhanced the rate of tumorigenesis induced by mammary-targeted Ha-Ras or c-Myc, enhanced gene expression profiles and immunohistochemical features of epithelial mesenchymal transition (EMT) and putative cancer stem cells in vivo. Silencing of p21(CIP1) enhanced, and expression of p21(CIP1) repressed, features of EMT in transformed immortal human MEC lines. p21(CIP1) attenuated oncogene-induced BT-IC and mammosphere formation. Thus, the in vitro cell culture assays reflect the changes observed in vivo in transgenic mice. These findings establish a link between the loss of p21(CIP1) and the acquisition of breast cancer EMT and stem cell properties in vivo.