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Biofumigation has been proposed as an alternative to soil fumigation to manage soil-borne diseases including potato early dying disease complex (PED). This study examined the potential of using brown mustard (Mustard juncea) biofumigation to manage PED under rain-fed potato production in New Brunswick, Canada in two trials between 2017 and 2020 in comparison with chloropicrin fumigation and a conventional barley rotation. Biofumigation increased yield in one trial, but not in a second trial where the potato crop experienced severe drought, whereas chloropicrin fumigation increased yield in both trials. Biofumigation was effective in suppressing root-lesion nematode (RLN, Pratylenchus spp.) counts in both trials, but was ineffective in suppressing V. dahliae population density. Chloropicrin fumigation was effective in suppressing RLN counts and V. dahliae population density only in the hill where injected, but the effect was short-lived as the population density of V. dahliae in the hill increased to the level of the control in one potato growing season. Biofumigation may be an alternative to chloropicrin fumigation in managing PED, particularly in fields with high RLN population but relatively low Verticillium population density. However, neither biofumigation nor fumigation used alone may be sustainable in the short-term potato rotations commonly used in New Brunswick, and additional beneficial practices are required to sustain productivity in the long-term.

Figure S1. Detection of miR-10b, miR-10a, Hoxd4, and Hoxd3. Figure S2. Body and organ weight. Figure S3. Overall survival and fertility. Figure S4. Complete blood cell count. Figure S5. Organ histology. Figure S6. Mammary tumor cell proliferation. Figure S7. PyMT antibody characterization. Figure S8. miR-10b target screen. Figure S9. TBX5 target identification. Figure S10. TBX5 inhibits breast cancer cell proliferation, migration, and invasion. Figure S11. Clinical correlation.

Legends for Supplementary Figures S1-S11

The invasive and metastatic properties of many human tumors have been associated with upregulation of the miRNA miR-10b, but its functional contributions in this setting have not been fully unraveled. Here, we report the generation of miR-10b–deficient mice, in which miR-10b is shown to be largely dispensable for normal development but critical to tumorigenesis. Loss of miR-10b delays oncogene-induced mammary tumorigenesis and suppresses epithelial–mesenchymal transition, intravasation, and metastasis in a mouse model of metastatic breast cancer. Among the target genes of miR-10b, the tumor suppressor genes Tbx5 and Pten and the metastasis suppressor gene Hoxd10 are significantly upregulated by miR-10b deletion. Mechanistically, miR-10b promotes breast cancer cell proliferation, migration, and invasion through inhibition of the expression of the transcription factor TBX5, leading to repression of the tumor suppressor genes DYRK1A and PTEN. In clinical specimens of breast cancer, the expression of TBX5, HOXD10, and DYRK1A correlates with relapse-free survival and overall survival outcomes in patients. Our results establish miR-10b as an oncomiR that drives metastasis, termed a metastamiR, and define the set of critical tumor suppressor mechanisms it overcomes to drive breast cancer progression. Cancer Res; 76(21); 6424–35. ©2016 AACR.

Additional details related to the Materials and Methods section that appears in the main text.

A survey on field pea (Pisum sativum) root rot was carried out in Prince Edward Island (PEI) and New Brunswick (NB), Canada, between July and August, 2018. The average disease incidence was 75% and 78%, and severity was 3.5 and 2.8 on a 1 to 7 scale for NB and PEI, respectively (Schneider and Kelly 2000). Symptoms included seedling stunting, root rot, and wilting. Surface sterilized diseased root segments were incubated on water agar at 25°C for 5 days. Pure isolates were obtained by single spore culturing. A total of 210 isolates were identified as Fusarium spp. Five isolates were identified as F. commune by morphological and molecular characteristics (Leslie and Summerell, 2006; Skovgaard et al. 2003). The isolates on Potato Dextrose Agar produced whitish fluffy mycelia on the upper surface and grayish yellow coloration on the bottom surface of the colony cultured at 25°C in darkness. The isolates on carnation leaf agar at 25°C in darkness formed abundant chlamydospores and macroconidia but rare microconidia with 0 to1 septa, measuring 6.2 to 12.5 x 2.7 to 3.6 µm (n = 5). Macroconidia were typically fusiform with a slightly curved apical cell and a foot-shaped basal cell, bending equally toward both ends. Three-septate conidia were 26.8 to 39.3 x 3.6 to 4.5 µm (n = 20) and five-septate conidia were 56.2 to 64.3 x 4.5 to 5.4 µm in size (n = 10). Chlamydospores were smooth, terminal, and single, 7 to 12.5 µm in diameter (n = 20). Genomic DNA of the five isolates were used to amplify and sequence the translation elongation factor 1α (TEF-1α) and the mitochondrial small subunit ribosomal DNA (mtSSU rDNA) using EF1/EF2 and NSM1/NSM2 primer pairs (White et al 1990; O’Donnell et al 2000), respectively, which were used to define the F. commune (Skovgaard et al. 2003). The mtSSU rDNA sequences were deposited in GenBank, OP752229 to OP752232 and OP752234 for isolates GR11-8, GR11-9, GR1-21, FRDC11-1 and FRDC11-2, respectively, and the TEF-1α sequences were assigned OP831956 to OP831959 and OP831961 for GR11-8, GR11-9, GR1-21, FRDC11-1, and FRDC11-2, respectively. The sequence similarities of the five isolates with ex holotype culture NRRL 31076 ranged from 98.69% to 99.79% for the TEF-1α (AF362263.1), with the matching base pairs of 526/533, 523/528, 483/484, 497/502 and 527/533, respectively, and 99.83 to 100% for the mtSSU rDNA (AF362279.1), with the matching base pairs of 633/634, 633/634, 600/601, 633/634, and 621/621, respectively. The sequences of mtSSU rDNA and TEF-1α for the F. commune type species and related Fusarium species were retrieved from NCBI. A phylogenetic tree constructed using the combined sequences of mtSSU rDNA and TEF-1α showed the five isolates clustered with F. commune. Three isolates (GR11-8, GR11-9, GR1-21) were used for pathogenicity testing with four replicates of four plants each and the trial was repeated twice. Seeds of field pea (CDC Limerick) were soaked in 2% sodium hypochlorite for 2 minutes, washed three times with sterilized distilled water, and then were soaked in conidial suspension at 2 × 106 conidia / mL or in sterilized distilled water as a control for 16 h in darkness at 20°C. Seeds were placed in sterilized vermiculite in a greenhouse at 24 / 18°C day / night temperature with a 16 h photoperiod. Three weeks after planting, the tested isolates were observed to cause seed decay, root rot, and seedling stunting, with disease severity ranging from 5 to 7 based on 1 to 7 scale in repeated trials. No symptoms were observed on the control plants. F. commune isolates were re-isolated and confirmed by sequencing the mtSSU rDNA and TEF-1α. F. commune was reported in Alberta causing soybean root rot (Zhou et al. 2018) but this is the first report of F. commune causing root rot of field pea in Canada. Considering its high pathogenicity in field pea and in soybean, the prevalence, host range and geographic distribution of this pathogen need further study.

In this study, a set of duplex reverse transcription PCR (RT-PCR)–mediated high-resolution DNA melting (HRM) analyses for simultaneous detection of potato mop-virus (PMTV) and its protist vector, Spongospora subterranea f. sp. subterranea (Sss), was developed. The infestation of soil by PMTV was detected with a tobacco-based baiting system. Total RNA extracted from the soil led to successful RT-PCR gel electrophoresis detection of both PMTV and Sss. To facilitate more efficient detection, newly designed primer pairs for PMTV RNA species (i.e., RNA-Rep, RNA-CP, and RNA-TGB) were analyzed together with the existing Sss primers via real-time RT-PCR. The resulting amplicons exhibited melting profiles that could be readily differentiated. Under duplex RT-PCR format, all PMTV and Sss primer combinations led to successful detection of respective PMTV RNA species and Sss in the samples by HRM analyses. When the duplex HRM assay was applied to soil samples collected from six fields at four different sites in New Brunswick, Canada, positive detection of PMTV or Sss was found in 63 to 100% samples collected from fields in which PMTV-infected tubers had been observed. In contrast, the samples from fields where neither PMTV- nor Sss-infected tubers had been observed resulted in negative detection by the assay. Bait tobacco bioassay for PMTV and Sss produced similar results. Of the soil samples collected from PMTV-infested fields, 63 to 83% and 100% led to PMTV and Sss infections in the bait tobacco plants, respectively, whereas no PMTV- or Sss-infected plants were obtained from soil samples collected from PMTV- and Sss-free fields.

Fusarium dry rot (FDR), caused by Fusarium species is an important potato disease world-wide and causes post-harvest rotting and seed-piece decay after planting. In this study, we compared two inoculation methods - cork borer wounding (CBW) and plastic screw wounding (PSW) – for consistent and reproducible disease development in potatoes. A plastic screw was used as a tool to make nearly identical wounds and simultaneously deliver similar quantities of inoculum into tubers, while at the same time sealing the wound in a single step. The PSW method resulted in tuber dry rot severities comparable to that obtained using the traditional CBW method. Tuber rot severities obtained using the two methods were highly correlated in the same sets of tubers, among the three inoculations conducted at different time periods (r values ranging from 0.56–0.95; p

To investigate the genotoxicity of metformin on planarian with different concentrations and exposure times.The planarians were treated, respectively, with 10 mmol/L and 50 mmol/L metformin for 1, 3, and 5 days, and then, the comet assay and random amplified polymorphic DNA (RAPD) analysis were performed. 13 random primers were used for PCR amplification with the genomic DNAs as templates. Planarians cultured in clear water were used as the control. Genomic template stability (GTS) was calculated by comparing and analyzing the RAPD patterns of the control group and the treatment groups.In the comet assay, DNA damage of planarians treated with 10 mmol/L metformin for 1, 3, and 5 days was 10.2%, 25.4%, and 36.8%, respectively, and that of planarians treated with 50 mmol/L metformin was 40.6%, 62.8%, and 65.4%, respectively. GTS values of planarians exposed to 10 mmol/L metformin for 1, 3, and 5 days were 64.1%, 62.8%, and 52.6%, respectively, and those of planarians exposed to 50 mmol/L metformin for 1, 3, and 5 days were 52.6%, 51.3%, and 50%, respectively. DNA damage increased and GTS values decreased with the increasing metformin exposure concentration and exposure time.Metformin has certain genotoxicity on planarian in a dose- and time-related manner. The comet assay and RAPD analysis are highly sensitive methods for detecting genotoxicity with drugs.

miR-219-5p has been reported to act as either a tumor suppressor or a tumor promoter in different cancers by targeting different genes. In the present study, we demonstrated that miR-219-5p negatively regulated the expression of TBXT, a known epithelial–mesenchymal transition (EMT) inducer, by directly binding to TBXT 3′-untranslated region. As a result of its inhibition on TBXT expression, miR-219-5p suppressed EMT and cell migration and invasion in breast cancer cells. The re-introduction of TBXT in miR-219-5p overexpressing cells decreased the inhibitory effects of miR-219 on EMT and cell migration and invasion. Moreover, miR-219-5p decreased breast cancer stem cell (CSC) marker genes expression and reduced the mammosphere forming capability of cells. Overall, our study highlighted that TBXT is a novel target of miR-219-5p. By suppressing TBXT, miR-219-5p plays an important role in EMT and cell migration and invasion of breast cancer cells.

In this study, a set of duplex reverse transcription PCR (RT-PCR)-mediated high-resolution DNA melting (HRM) analyses for simultaneous detection of potato mop-virus (PMTV) and its protist vector

Background Anthocyanins (ACNs) are capable of suppressing breast cancer growth; however, investigation on the effect and mechanism of ACNs on epithelial-to-mesenchymal transition (EMT), and cell migration and invasion in breast cancer cells is limited. A complete understanding of those properties may provide useful information on of how to use these natural compounds for cancer prevention and treatment. Objectives The aim of this work was to investigate the role of cyanidin-3-O-glucoside (Cy3G), one of the most widely distributed ACNs in edible fruits, in the EMT process, and cell migration and invasion of breast cancer cells, and its underlying molecular mechanisms of how Cy3G establishes these functional roles in these cells. Methods MDA-MB-231 and MDA-MB-468 breast cancer cells were treated with Cy3G (20 μM) for 24 h, and then the cells were used for cell migration and invasion assay. Western blotting, luciferase assay, ubiquitination assay, gene knockdown, and cycloheximide chase assay were performed to analyze the molecular mechanisms of Cy3G in suppressing EMT, and cell migration and invasion. Results Cy3G inhibited the EMT process in these cells and significantly suppressed the migration and invasion of breast cancer cells (P ≤ 0.05) by upregulating Krüppel-like factor 4 (KLF4) expression at protein level. KLF4 knockdown in MDA-MB-231 cells did not reveal any change in EMT marker expression, and cell migration and invasion upon treatment with Cy3G (P ≥ 0.05), which strongly indicated that the effects of Cy3G were mediated by KLF4. Furthermore, we determined that Cy3G indirectly upregulated KLF4 expression by downregulating FBXO32, which is the E3 ligase of KLF4. Conclusion Cy3G is a potential anticancer reagent as it can inhibit EMT and breast cancer cell migration and invasion by upregulating KLF4.

The invasive and metastatic properties of many human tumors have been associated with upregulation of the miRNA miR-10b, but its functional contributions in this setting have not been fully unraveled. Here, we report the generation of miR-10b–deficient mice, in which miR-10b is shown to be largely dispensable for normal development but critical to tumorigenesis. Loss of miR-10b delays oncogene-induced mammary tumorigenesis and suppresses epithelial–mesenchymal transition, intravasation, and metastasis in a mouse model of metastatic breast cancer. Among the target genes of miR-10b, the tumor suppressor genes Tbx5 and Pten and the metastasis suppressor gene Hoxd10 are significantly upregulated by miR-10b deletion. Mechanistically, miR-10b promotes breast cancer cell proliferation, migration, and invasion through inhibition of the expression of the transcription factor TBX5, leading to repression of the tumor suppressor genes DYRK1A and PTEN. In clinical specimens of breast cancer, the expression of TBX5, HOXD10, and DYRK1A correlates with relapse-free survival and overall survival outcomes in patients. Our results establish miR-10b as an oncomiR that drives metastasis, termed a metastamiR, and define the set of critical tumor suppressor mechanisms it overcomes to drive breast cancer progression. Cancer Res; 76(21); 6424–35. ©2016 AACR.

In plants, transcriptional regulation is the most important tool for modulating flavonoid biosynthesis. The basic helix-loop-helix transcription factors are only one example how then flavonoid pathway is regulated. There are other transcription factors as well. In this study, the codon-optimized VvbHLH1 gene from grape was chemically synthesized. Overexpression of VvbHLH1 significantly increased the accumulation of flavonoids and enhanced salt and drought tolerance in transgenic Arabidopsis thaliana plants. Real-time quantitative PCR analysis showed that overexpression of VvbHLH1 resulted in the up-regulation of genes involved in flavonoid biosynthesis, abscisic acid (ABA) signaling pathway, proline biosynthesis, stress responses and ROS scavenging under salt and drought stresses. Further analyses under salt and drought stresses showed significant increases of ABA and proline content, superoxide dismutase and peroxidase activities, as well as significant reduction of hydrogen peroxide (H2O2) and malonaldehyde content. The results demonstrate the explicit role of VvbHLH1 in conferring salt and drought tolerance by increasing the accumulation of flavonoids and ABA signalling in transgenic A. thaliana. The VvbHLH1 gene has the potential to be used to increase the content of valuable flavonoids and improve the tolerance to abiotic stresses in A. thaliana and other plants.

SummaryMolecular mechanisms underpinning nonalcoholic fatty liver disease (NAFLD) are not well understood. The earliest step of NAFLD is hepatic steatosis, which is one of the main characteristics of aging liver. Here, we present a molecular scenario of age-related liver steatosis. We show that C/EBPα-S193D knockin mice have age-associated epigenetic changes and develop hepatic steatosis at 2 months of age. The underlying mechanism of the hepatic steatosis in old wild-type (WT) mice and in young S193D mice includes increased amounts of tripartite p300-C/EBPα/β complexes that activate promoters of five genes that drive triglyceride synthesis. Knockdown of p300 in old WT mice inhibits hepatic steatosis. Indeed, transgenic mice expressing dominant-negative p300 have fewer C/EBPα/β-p300 complexes and do not develop age-dependent hepatic steatosis. Notably, the p300-C/EBPα/β pathway is activated in the livers of patients with NAFLD. Thus, our results show that p300 and C/EBP proteins are essential participants in hepatic steatosis.

The transcriptional co-regulator SKI is a potent inhibitor of TGFbeta-growth inhibitory signals. SKI binds to receptor-activated Smads in the nucleus, forming repressor complexes containing HDACs, mSin3, NCoR, and other protein partners. Alternatively, SKI binds to activated Smads in the cytoplasm, preventing their nuclear translocation. SKI is necessary for anchorage-independent growth of melanoma cells in vitro, and most important, for human melanoma xenograft growth in vivo. We recently identified a novel role of SKI in TGFbeta signaling. SKI promotes the switch of Smad3 from repressor of proliferation to activator of oncogenesis by facilitating phosphorylations in the linker domain. High levels of endogenous SKI are required by the tumor promoting trait of TGFbeta to induce expression of the plasminogen-activator inhibitor-1 (PAI-1), sustained expression of C-Myc and for aborting upregulation of p21(Waf-1). Here we discuss how SKI diversifies and amplifies its functions by associating with multiple protein partners and by promoting Smad3 linker phosphorylation(s) in response to TGFbeta signaling in melanoma cells.

The SKI protein represses the TGF-beta tumor suppressor pathway by associating with the Smad transcription factors. SKI is upregulated in human malignant melanoma tumors in a disease-progression manner and its overexpression promotes proliferation and migration of melanoma cells in vitro. The mechanisms by which SKI antagonizes TGF-beta signaling in vivo have not been fully elucidated. Here we show that human melanoma cells in which endogenous SKI expression was knocked down by RNAi produced minimal orthotopic tumor xenograft nodules that displayed low mitotic rate and prominent apoptosis. These minute tumors exhibited critical signatures of active TGF-beta signaling including high levels of nuclear Smad3 and p21(Waf-1), which are not found in the parental melanomas. To understand how SKI promotes tumor growth we used gain- and loss-of-function approaches and found that simultaneously to blocking the TGF-beta-growth inhibitory pathway, SKI promotes the switch of Smad3 from tumor suppression to oncogenesis by favoring phosphorylations of the Smad3 linker region in melanoma cells but not in normal human melanocytes. In this context, SKI is required for preventing TGF-beta-mediated downregulation of the oncogenic protein c-MYC, and for inducing the plasminogen activator inhibitor-1, a mediator of tumor growth and angiogenesis. Together, the results indicate that SKI exploits multiple regulatory levels of the TGF-beta pathway and its deficiency restores TGF-beta tumor suppressor and apoptotic activities in spite of the likely presence of oncogenic mutations in melanoma tumors.

The proteins SKI and SnoN are implicated in processes as diverse as differentiation, transformation and tumor progression. Until recently, SKI was solely viewed as a nuclear protein with a principal function of inhibiting TGF-beta signaling through its association with the Smad proteins. However, new studies suggest that SKI plays additional roles not only inside but also outside the nucleus. In normal melanocytes and primary non-invasive melanomas, SKI localizes predominantly in the nucleus, whereas in primary invasive melanomas SKI displays both nuclear and cytoplasmic localization. Intriguingly, metastatic melanoma tumors display nuclear and cytoplasmic or predominantly cytoplasmic SKI distribution. Cytoplasmic SKI is functional, as it associates with Smad3 and prevents its nuclear localization mediated by TGF-beta. SKI can also function as a transcriptional activator, targeting the beta -catenin pathway and activating MITF and NrCAM, two proteins involved in survival, migration and invasion. Intriguingly, SKI appears to live a dual life, one as a tumor suppressor and another as a transforming protein. Loss of one copy of mouse ski increases susceptibility to tumorigenesis in mice, whereas its overexpression is associated with cancer progression of human melanoma, esophageal, breast and colon. The molecular reasons for such dramatic change in SKI function appear to result from new acquired activities. In this review, we discuss the mechanisms by which SKI regulates crucial pathways involved in the progression of human malignant melanoma.

In rodents, the steroid hormone estrogen (E) profoundly influences the early events in the uterus leading to embryo implantation. It is thought that E triggers the expression of a unique set of genes in the endometrium that in turn control implantation. To identify these E-induced genes, we employed a delayed implantation model system in which embryo attachment to rat endometrium is dependent upon E administration. Using a gene expression screen method, we isolated a number of cDNAs representing mRNAs whose expression is either turned on or turned off in response to an implantation-inducing dose of E. We identified one of these cDNAs as that encoding secretory leukocyte protease inhibitor (SLPI), an inhibitor of serine proteases. The expression of SLPI mRNA was induced in the uteri of ovariectomized rats in response to E, confirming the hormonal regulation of this molecule. Spatiotemporal analysis revealed a biphasic pattern of expression of SLPI mRNA during early pregnancy. A considerable amount of SLPI mRNA was detected in the uterine epithelium on Day 1 of pregnancy. The level of this mRNA, however, declined sharply on Days 2 and 3 of gestation. Interestingly, on Day 4 of gestation, there was a marked resurgence in SLPI mRNA expression in the uterine epithelium. This second burst of SLPI expression diminished by Day 6 of pregnancy. The transient induction of SLPI mRNA during Days 4 and 5 overlapped with the window of implantation in the rat. Although the precise function of SLPI in the uterus eludes us presently, its known effects as a serine protease inhibitor in other tissues and its hormone-induced expression in the rat uterus immediately preceding implantation lead us to propose that this gene plays an important role in controlling excessive proteolysis and inflammation during a critical phase of early pregnancy. estradiol receptor, female reproductive tract, implantation, uterus

Tumour cells associated with therapy resistance (radioresistance and drug resistance) are likely to give rise to local recurrence and distant metastatic relapse. Recent studies revealed microRNA (miRNA)-mediated regulation of metastasis and epithelial-mesenchymal transition; however, whether specific miRNAs regulate tumour radioresistance and can be exploited as radiosensitizing agents remains unclear. Here we find that miR-205 promotes radiosensitivity and is downregulated in radioresistant subpopulations of breast cancer cells, and that loss of miR-205 is highly associated with poor distant relapse-free survival in breast cancer patients. Notably, therapeutic delivery of miR-205 mimics via nanoliposomes can sensitize the tumour to radiation in a xenograft model. Mechanistically, radiation suppresses miR-205 expression through ataxia telangiectasia mutated (ATM) and zinc finger E-box binding homeobox 1 (ZEB1). Moreover, miR-205 inhibits DNA damage repair by targeting ZEB1 and the ubiquitin-conjugating enzyme Ubc13. These findings identify miR-205 as a radiosensitizing miRNA and reveal a new therapeutic strategy for radioresistant tumours.

Implantation of the developing blastocyst is regulated by multiple effectors, such as steroid hormones, growth factors, and cytokines. To understand how these diverse signaling pathways interact to modulate uterine gene expression, we employed a gene expression screen technique to identify the molecules that are induced in the periimplantation rat uterus. Here we report the isolation of a complementary DNA representing a novel gene, interferon-regulated gene 1 (IRG1). This gene exhibits significant homology to interferon (IFN)-alpha/beta-inducible human genes p27 and 6-16, indicating that these genes may belong to the same family. Consistent with this finding, expression of IRG1 messenger RNA (mRNA) in rat uterus increased about 20-fold in response to IFNalpha. Uterine expression of IRG1 was also stimulated by estrogen and was partially inhibited by an antiestrogen, ICI 182,780. In pregnant rats, IRG1 expression was high on day 1, but declined on days 2 and 3. The level of IRG1 mRNA again rose transiently on day 4 immediately preceding implantation. In situ hybridization analysis localized the IRG1 mRNA expression in the endometrial epithelium and the surrounding stroma. Interestingly, the expression of p27, which shows high homology to IRG1, was strongly enhanced in human endometrium during the midsecretory phase of the menstrual cycle, overlapping the putative window of implantation. Both IRG1 and p27 mRNAs are therefore induced in the endometrium in an implantation stage-specific manner. We also observed a synergistic interaction between IFNalpha and estrogen receptor signaling pathways that led to maximal induction of p27 mRNA in Ishikawa cells. Although the functional roles of IRG1 and p27 remain unclear, we describe for the first time, identification of a gene family regulated by IFNalpha in both rodent and human uteri. More importantly, our studies reveal that a complex interplay between the steroid hormone and IFN pathways regulates the expression of these genes in the endometrium at the time of implantation.

The steroid hormone estrogen profoundly influences the early events in the uterus leading to embryo implantation. It is thought that estrogen triggers the expression of a unique set of genes in the preimplantation endometrium that in turn control implantation. To identify these estrogen-induced genes, we used a delayed implantation model system in which embryo attachment to endometrium is dependent on estrogen administration. Using a mRNA differential display (DD) method, we isolated a number of cDNAs representing mRNAs whose expression is either turned on or turned off in response to an implantation-inducing dose of estrogen. We identified one of these cDNAs as that encoding rab11, a p21ras-like GTP-binding protein (G protein), which functions in the targeting of transport vesicles to the plasma membrane. In normal pregnant rats, rab11 mRNA was expressed at low levels on days 1–2 of pregnancy, but its expression was markedly enhanced (∼6- to 8-fold) between days 3–5 immediately before implantation. In situ hybridization and immunocytochemistry revealed that rab11 expression in the uterus was predominantly in the glandular epithelium. In ovariectomized rats, the expression of rab11 mRNA was induced in the endometrium in response to estrogen. To determine whether this effect of estrogen was mediated through its nuclear receptors, we examined rab11 expression in a transformed endometrial cell line, Ishikawa. In transient transfection experiments, we observed that overexpression of estrogen receptor (ER) α or β induced endogenous rab11 mRNA in a hormone-dependent manner. ER bound to an antagonist, ICI 182,780, failed to activate this gene expression. These findings, together with the observation that ERα but not ERβ is detected in the glands of the preimplantation uterus, indicate that rab11 is one of the proteins that are specifically induced by estrogen-complexed ERα in rat endometrium at the onset of implantation. Our results imply that estrogen, which induces the synthesis of many growth factors and their receptors and other secretory proteins that are thought to be critical for implantation, may also facilitate their transport to the membrane and/or secretion by stimulating the expression of rab11, a component of the membrane-trafficking pathway. This study therefore provides novel insights into the diverse cellular mechanisms by which estrogen, acting via its nuclear receptors, may influence blastocyst implantation.

Whether epithelial-mesenchymal transition (EMT) is always linked to increased tumorigenicity is controversial. Through microRNA (miRNA) expression profiling of mammary epithelial cells overexpressing Twist, Snail or ZEB1, we identified miR-100 as a novel EMT inducer. Surprisingly, miR-100 inhibits the tumorigenicity, motility and invasiveness of mammary tumor cells, and is commonly downregulated in human breast cancer due to hypermethylation of its host gene MIR100HG. The EMT-inducing and tumor-suppressing effects of miR-100 are mediated by distinct targets. While miR-100 downregulates E-cadherin by targeting SMARCA5, a regulator of CDH1 promoter methylation, this miRNA suppresses tumorigenesis, cell movement and invasion in vitro and in vivo through direct targeting of HOXA1, a gene that is both oncogenic and pro-invasive, leading to repression of multiple HOXA1 downstream targets involved in oncogenesis and invasiveness. These findings provide a proof-of-principle that EMT and tumorigenicity are not always associated and that certain EMT inducers can inhibit tumorigenesis, migration and invasion., Author Summary Induction of epithelial-mesenchymal transition (EMT) in epithelial tumor cells has been shown to enhance migration, invasion and cancer ‘stemness’. Here we demonstrate that a miRNA downregulated in human breast tumors, miR-100, can simultaneously induce EMT and inhibit tumorigenesis, migration and invasion through direct targeting of distinct genes. This is the first report of an EMT inducer that suppresses cell movement and tumor invasion, which indicates that EMT is not always associated with increased tumorigenesis, migration and invasion, and that all EMT inducers are not equal: while some of them can promote tumorigenicity, motility and invasiveness, others inhibit these properties owing to their ability to concurrently target both EMT-repressing genes and oncogenic/pro-invasive genes. These findings provide new insights into the complex roles of EMT inducers.

The tumour suppressor PTEN is frequently lost in human cancers. In addition to gene mutations and deletions, recent studies have revealed the importance of post-translational modifications, such as ubiquitylation, in the regulation of PTEN stability, activity and localization. However, the deubiquitylase that regulates PTEN polyubiquitylation and protein stability remains unknown. Here we screened a total of 30 deubiquitylating enzymes (DUBs) and identified five DUBs that physically associate with PTEN. One of them, USP13, stabilizes the PTEN protein through direct binding and deubiquitylation of PTEN. Loss of USP13 in breast cancer cells promotes AKT phosphorylation, cell proliferation, anchorage-independent growth, glycolysis and tumour growth through downregulation of PTEN. Conversely, overexpression of USP13 suppresses tumorigenesis and glycolysis in PTEN-positive but not PTEN-null breast cancer cells. Importantly, USP13 protein is downregulated in human breast tumours and correlates with PTEN protein levels. These findings identify USP13 as a tumour-suppressing protein that functions through deubiquitylation and stabilization of PTEN.

There is a pressing need to identify prognostic markers of metastatic disease and targets for treatment. Combining high-throughput RNA sequencing, functional characterization, mechanistic studies and clinical validation, we identify leukemia inhibitory factor receptor (LIFR) as a breast cancer metastasis suppressor downstream of the microRNA miR-9 and upstream of Hippo signaling. Restoring LIFR expression in highly malignant tumor cells suppresses metastasis by triggering a Hippo kinase cascade that leads to phosphorylation, cytoplasmic retention and functional inactivation of the transcriptional coactivator YES-associated protein (YAP). Conversely, loss of LIFR in nonmetastatic breast cancer cells induces migration, invasion and metastatic colonization through activation of YAP. LIFR is downregulated in human breast carcinomas and inversely correlates with metastasis. Notably, in approximately 1,000 nonmetastatic breast tumors, LIFR expression status correlated with metastasis-free, recurrence-free and overall survival outcomes in the patients. These findings identify LIFR as a metastasis suppressor that functions through the Hippo-YAP pathway and has significant prognostic power.

Hepatic cytochrome P450 (CYP) genes, including members of CYP1 to CYP4 families, comprise the majority of the CYP gene superfamily. Previous study has demonstrated that HepG2-specific transcriptional activation of two CYP2C genes was dependent on a common element that bound a HepG2 nuclear protein designated HPF-1 (Venepally, P., Chen, D., and Kemper, B. (1992) J. Biol. Chem. 267, 17333-17338). This cis-acting element is highly homologous to the hepatocyte nuclear factor 4 (HNF-4) binding motif and is present in the promoters of more than 20 other CYP2 genes. To investigate the relationship between HPF-1 and HNF-4, we have compared their tissue distribution, DNA binding, and immunochemical characteristics, as well as transcriptional activity of their recognition elements. DNase I footprint analyses and gel-shift assays indicated that HPF-1, like HNF-4, was present in liver and kidney, but not brain and spleen. Both factors bound to either the HPF-1 site in the CYP2C2 promoter or an HNF-4 site in the human apolipoprotein CIII promoter. These complexes could be "supershifted" by an antiserum specific for HNF-4. When the sequence of the HPF-1 site in the CYP2C2 promoter was changed to that of the apolipoprotein CIII HNF-4 site, comparable transcriptional activities were obtained with the wild-type promoter and the HNF-4 mutant in transfected HepG2 cells. Cotransfection of HNF-4 with CYP2C2 promoter-luciferase constructs in COS-1 cells indicated that HNF-4 could trans-activate the promoters containing the HPF-1 site. These results indicate that the HPF-1 motif is a functional HNF-4-binding site, and the common immunological properties indicate that HPF-1 and HNF-4 are closely related and possibly identical. HNF-4, therefore, may act as a common regulator for the liver-specific expression of many CYP2 genes.

To analyze the transcriptional regulatory elements in rabbit cytochrome P450IIC genes, varying lengths of the 5'-flanking regions of CYP2C1 and CYP2C2 were fused to a luciferase reporter gene. Promoter activity was assayed by transfection into HepG2 cells, a hepatic cell line, and monkey kidney COS-1 cells, a nonhepatic cell line. Activity of the CYP2C1 promoter in HepG2 cells increased slightly with progressive 5' deletions of the 5'-flanking region from nucleotide -3600 to -1500 relative to the transcription start site. Additional deletions to -900, -358, and -116 each reduced activity by about 50%, and deletion of the sequence from -116 to -67 reduced activity by a factor of 12. Activity of the CYP2C2 promoter increased about 3-fold with progressive 5' deletions of sequence from nucleotide -3500 to -410. In contrast, deletions of sequences from -251 to -193 and from -133 to -64 reduced promoter activity by factors of 2 and 8, respectively. In COS-1 cells, the maximum activities of the CYP2C1 and CYP2C2 promoters normalized to a Rous sarcoma viral promoter were about 10-20% of that in the HepG2 cells. The changes in activity between different constructions in COS-1 cells largely paralleled those in the HepG2 cells except for deletions of the sequences -133 to -64 and -116 to -67 for CYP2C1 and CYP2C2, respectively, which produced the largest reduction of promoter activity in HepG2 cells but had little effect in COS-1 cells. These results show that HepG2-specific regulatory elements are present in the regions between -120 and -65 in both genes. Nuclear proteins from HepG2 cells, but not from COS-1 cells, bound to sequences within these regions, and the binding was inhibited by an oligonucleotide containing a sequence conserved in rabbit P450IIC genes which has been designated the HepG2-specific P450 2C factor-1 (HPF1) motif. Mutation of this sequence eliminated the binding of nuclear proteins and reduced transcriptional activity 25-fold. The HPF1 binding sequence is conserved in CYP2A, CYP2C, and CYP2D genes and resembles the binding motif for hepatic nuclear factor-4. These results demonstrate that CYP2C1 and CYP2C2 contain several potential regulatory elements for basal expression, including one HepG2-specific sequence that may be important for liver expression.

Cellular localization, association with different protein partners, and posttranslational modifications can dramatically change protein function. SKI and the highly homologous protein snoN are potent repressors of transforming growth factor-β signaling through their association with the Smad proteins. In fact, SKI can act as molecular switch converting the Smad proteins from an activating to a repressing entity on chromatin. SKI also plays additional roles in melanomas: in association with the LIM protein FHL2 activates β-catenin signaling, a pathway associated with cancer progression. This chapter reviews the transcriptional co-repressor and co-activator activities of SKI and discusses their biological significance for melanoma tumor progression.

The retinoblastoma (RB)/p16(INK4a) pathway regulates senescence of human melanocytes in culture and oncogene-induced senescence of melanocytic nevi in vivo. This senescence response is likely due to chromatin modifications because RB complexes from senescent melanocytes contain increased levels of histone deacetylase (HDAC) activity and tethered HDAC1. Here we show that HDAC1 is prominently detected in p16(INK4a)-positive, senescent intradermal melanocytic nevi but not in proliferating, recurrent nevus cells that localize to the epidermal/dermal junction. To assess the role of HDAC1 in the senescence of melanocytes and nevi, we used tetracycline-based inducible expression systems in cultured melanocytic cells. We found that HDAC1 drives a sequential and cooperative activity of chromatin remodeling effectors, including transient recruitment of Brahma (Brm1) into RB/HDAC1 mega-complexes, formation of heterochromatin protein 1 beta (HP1 beta)/SUV39H1 foci, methylation of H3-K9, stable association of RB with chromatin and significant global heterochromatinization. These chromatin changes coincide with expression of typical markers of senescence, including the senescent-associated beta-galactosidase marker. Notably, formation of RB/HP1 beta foci and early tethering of RB to chromatin depends on intact Brm1 ATPase activity. As cells reached senescence, ejection of Brm1 from chromatin coincided with its dissociation from HP1 beta/RB and relocalization to protein complexes of lower molecular weight. These results provide new insights into the role of the RB pathway in regulating cellular senescence and implicate HDAC1 as a likely mediator of early chromatin remodeling events.

Overexpression of the oncoprotein SKI correlates with the progression of human melanoma in vivo. SKI is known to curtail the growth inhibitory activity of tumor growth factor beta through the formation of repressive transcriptional complexes with Smad2 and Smad3 at the p21(Waf-1) promoter. Here, we show that SKI also stimulates growth by activating the Wnt signaling pathway. From a yeast two-hybrid screen and immunoprecipitation studies, we identified the protein FHL2/DRAL as a novel SKI binding partner. FHL2, a LIM-only protein, binds beta-catenin and can function as either a transcriptional repressor or activator of the Wnt signaling pathway. SKI enhanced the activation of FHL2 and/or beta-catenin- regulated gene promoters in melanoma cells. Among the SKI targets were microphthalmia-associated transcription factor and Nr-CAM, two proteins associated with melanoma cell survival, growth, motility, and transformation. Transient overexpression of SKI and FHL2 in ski(-/-) melanocytes synergistically enhanced cell growth, and stable overexpression of SKI in a poorly clonogenic human melanoma cell line was sufficient to stimulate rapid proliferation, decreasing the number of cells in the G(1) phase of the cell cycle, and dramatically increasing clonogenicity, colony size and motility. Taken together, these results suggest that by targeting members of the tumor growth factor beta and beta-catenin pathways, SKI regulates crucial events required for melanoma growth, survival, and invasion.

The steroid hormone estrogen profoundly influences growth and differentiation programs in the reproductive tract of cycling and pregnant mamals. It is thought that estrogen exerts its cellular effects by regulating the expression of specific target genes. We utilized a messenger RNA differential display method to identify the genes whose expression is modulated by estrogen in the preimplantation rat uterus. Here we report the cloning of a novel gene (ERG1) that is tightly regulated by estrogen in two key reproductive tissues, the uterus and oviduct. Spatio-temporal analyses reveal that ERG1 mRNA is expressed in a highly stage-specific manner in the uterus and oviduct, and its expression is restricted to the surface epithelium of both of these tissues. Nucleotide sequence analysis of the full-length ERG1 cDNA indicates that it has an open reading frame of 1821 nuceotides encoding a putative protein of 607 amino acids with a single transmembrane domain and a short cytoplasmic tail. The extracellular part of the protein contains several distinct structural motifs. These include a zona pellucida binding domain, which is present in a number of proteins such as the zona pellucida sperm binding proteins, and uromodulin, In addition, there is a repeat of a motif called CUB domain, which exists in a number of genes involved in development and differentiation such as bone morphogenetic protein 1 (BMP1). Although the precise function of ERG1 eludes us presently, its unique pattern of expression in the uterus and oviduct and its regulation by estrogen, a principal reproductive hormone, lead us to speculate that this novel gene plays an important role in events during the reproductive cycle and early pregnancy.