The Anoikis Effector Bit1 Displays Tumor Suppressive Function in Lung Cancer Cells.

The mitochondrial Bit1 (Bcl-2 inhibitor of transcription 1) protein is a part of an apoptotic pathway that is uniquely regulated by integrin-mediated attachment. As an anoikis effector, Bit1 is released into the cytoplasm following loss of cell attachment and induces a caspase-independent form of apoptosis. Considering that anoikis resistance is a critical determinant of transformation, we hypothesized that cancer cells may circumvent the Bit1 apoptotic pathway to attain anchorageindependence and tumorigenic potential. Here, we provide the first evidence of the tumor suppressive effect of Bit1 through a mechanism involving anoikis induction in human lung adenocarcinoma derived A549 cells. Restitution of Bit1 in anoikis resistant A549 cells is sufficient to induce detachment induced-apoptosis despite defect in caspase activation and impairs their anchorage-independent growth. Conversely, stable downregulation of Bit1 in these cells significantly enhances their anoikis resistance and anchorage-independent growth. The Bit1 knockdown cells exhibit significantly enhanced tumorigenecity in vivo. It has been previously shown that the nuclear TLE1 corepressor is a putative oncogene in lung cancer, and we show here that TLE1 blocks Bit1 mediated anoikis in part by sequestering the pro-apoptotic partner of Bit1, the Amino-terminal Enhancer of Split (AES) protein, in the nucleus. Taken together, these findings suggest a tumor suppressive role of the caspase-independent anoikis effector Bit1 in lung cancer. Consistent with its role as a tumor suppressor, we have found that Bit1 is downregulated in human non-small cell lung cancer (NSCLC) tissues

Secondary Metabolic Gene Cluster Silencing in Aspergillus Nidulans

In contrast to most primary metabolism genes, the genes involved in secondary metabolism and certain nutrient utilization pathways are clustered in fungi. Recently a nuclear protein, LaeA, was found to be required for the transcription of several secondary metabolite gene clusters in Aspergillus nidulans. Here we show that LaeA regulation does not extend to nutrient utilization or the spoC1 sporulation clusters. One of the secondary metabolite clusters regulated by LaeA contains the positive regulatory (i.e. aflR) and biosynthetic genes required for biosynthesis of sterigmatocystin (ST), a carcinogenic toxin. Analysis of ST gene cluster expression indicates LaeA regulation of the cluster is location specific as transcription of genes bordering the ST cluster are unaffected in a ΔlaeA mutant and placement of a primary metabolic gene, argB, in the ST cluster resulted in argB silencing in the ΔlaeA background. ST cluster gene expression was remediated when an additional copy of aflR was placed outside of the cluster but not when placed in the cluster. Site-specific mutation of an s-adenosyl methionine (AdoMet) binding site in LaeA generated a ΔlaeA phenotype suggesting the protein to be a methyltransferase

Metastasis of Tumor Cells Is Enhanced by Downregulation of Bit1

Resistance to anoikis, which is defined as apoptosis induced by loss of integrin-mediated cell attachment to the extracellular matrix, is a determinant of tumor progression and metastasis. We have previously identified the mitochondrial Bit1 (Bcl-2 inhibitor of transcription) protein as a novel anoikis effector whose apoptotic function is independent from caspases and is uniquely controlled by integrins. In this report, we examined the possibility that Bit1 is suppressed during tumor progression and that Bit1 downregulation may play a role in tumor metastasis.Using a human breast tumor tissue array, we found that Bit1 expression is suppressed in a significant fraction of advanced stages of breast cancer. Targeted disruption of Bit1 via shRNA technology in lowly aggressive MCF7 cells conferred enhanced anoikis resistance, adhesive and migratory potential, which correlated with an increase in active Extracellular kinase regulated (Erk) levels and a decrease in Erk-directed phosphatase activity. These pro-metastasis phenotypes were also observed following downregulation of endogenous Bit1 in Hela and B16F1 cancer cell lines. The enhanced migratory and adhesive potential of Bit1 knockdown cells is in part dependent on their high level of Erk activation since down-regulating Erk in these cells attenuated their enhanced motility and adhesive properties. The Bit1 knockdown pools also showed a statistically highly significant increase in experimental lung metastasis, with no differences in tumor growth relative to control clones in vivo using a BALB/c nude mouse model system. Importantly, the pulmonary metastases of Bit1 knockdown cells exhibited increased phospho-Erk staining.These findings indicate that downregulation of Bit1 conferred cancer cells with enhanced anoikis resistance, adhesive and migratory properties in vitro and specifically potentiated tumor metastasis in vivo. These results underscore the therapeutic importance of restoring Bit1 expression in cancer cells to circumvent metastasis at least in part through inhibition of the Erk pathway

The L1 Retrotranspositional Stimulation by Particulate and Soluble Cadmium Exposure is Independent of the Generation of DNA Breaks.

Human exposure to toxic metals is a concern of the highest priority, due to their vast array of biological effects, including carcinogenicity. The particulate (water insoluble) form of several heavy metals presents a higher carcinogenic potential than its soluble counterparts. Our previous work demonstrates that the particulate forms of different heavy metals, such as nickel oxide, cadmium sulfide and mercury sulfide, stimulate human L1 mobile element activity leading to genomic instability. We present data demonstrating that the soluble form of CdCl2 also stimulates L1 retrotransposition in a dose-dependent manner comparable to the insoluble carcinogenic form of this compound. Reproducible results demonstrated a 2 to 3 fold dose-dependent increase in L1 retrotransposition compared to control cells. Heavy metals may cause DNA breaks through the generation of reactive oxygen species. However, evaluation of DNA damage by comet assay revealed no differences between the negative controls and the CdS-treated cells. In addition, active L1 elements express a protein with endonuclease activity that can generate toxicity through the creation of double strand breaks. To determine the contribution of the L1 endonuclease to the toxicity observed in our metal treatment assays, we compared the wild-type L1 vector with an L1 endonuclease-mutant vector. The presence of an active L1 endonuclease did not contribute significantly to the toxicity observed in any of the CdCl2 or CdS doses evaluated. No correlation between the creation of DNA breaks and L1 activity was observed. Alternatively, heavy metals inhibit enzymatic reactions by displacement of cofactors such as Zn and Mg from enzymes. Concomitant treatment with Mg(Ac)2 and Zn(Ac)2 ppb suppresses the stimulatory effect on L1 activity induced by the 3.8 ppb CdS treatment. Overall, these results are consistent with our previous observations, suggesting that the mechanism of L1 stimulation by heavy metals is most likely due to an overall inhibition of DNA repair proteins or other enzymes caused by the displacement of Mg and Zn from cellular proteins

The L1 Retrotranspositional Stimulation by Particulate and Soluble Cadmium Exposure is Independent of the Generation of DNA Breaks.

Human exposure to toxic metals is a concern of the highest priority, due to their vast array of biological effects, including carcinogenicity. The particulate (water insoluble) form of several heavy metals presents a higher carcinogenic potential than its soluble counterparts. Our previous work demonstrates that the particulate forms of different heavy metals, such as nickel oxide, cadmium sulfide and mercury sulfide, stimulate human L1 mobile element activity leading to genomic instability. We present data demonstrating that the soluble form of CdCl2 also stimulates L1 retrotransposition in a dose-dependent manner comparable to the insoluble carcinogenic form of this compound. Reproducible results demonstrated a 2 to 3 fold dose-dependent increase in L1 retrotransposition compared to control cells. Heavy metals may cause DNA breaks through the generation of reactive oxygen species. However, evaluation of DNA damage by comet assay revealed no differences between the negative controls and the CdS-treated cells. In addition, active L1 elements express a protein with endonuclease activity that can generate toxicity through the creation of double strand breaks. To determine the contribution of the L1 endonuclease to the toxicity observed in our metal treatment assays, we compared the wild-type L1 vector with an L1 endonuclease-mutant vector. The presence of an active L1 endonuclease did not contribute significantly to the toxicity observed in any of the CdCl2 or CdS doses evaluated. No correlation between the creation of DNA breaks and L1 activity was observed. Alternatively, heavy metals inhibit enzymatic reactions by displacement of cofactors such as Zn and Mg from enzymes. Concomitant treatment with Mg(Ac)2 and Zn(Ac)2 ppb suppresses the stimulatory effect on L1 activity induced by the 3.8 ppb CdS treatment. Overall, these results are consistent with our previous observations, suggesting that the mechanism of L1 stimulation by heavy metals is most likely due to an overall inhibition of DNA repair proteins or other enzymes caused by the displacement of Mg and Zn from cellular proteins

Heavy Metals Stimulate Human LINE-1 Retrotransposition.

L1 and Alu elements are among the most active retroposons (mobile elements) in the human genome. Several human diseases, including certain forms of breast cancer and leukemia, are associated with L1 and Alu insertions in functionally important areas of the genome. We present data demonstrating that environmental pollutants, such as heavy metals, can stimulate L1 retrotransposition in a tissue culture system using two different types of assays. The response to these agents was equivalent when using a cell line with a stably integrated L1 vector (genomic) or a by introducing the L1 vector by transient transfection (episomal) of the cell. Reproducible results showed that mercury (HgS), cadmium (CdS), and nickel (NiO) increase the activity of L1 by an average of three (3) fold p\u3c0.001. This observation is the first to link several carcinogenic agents with the increased retrotransposition activity of L1 as an alternate mechanism of generating genomic instability contributing to the process of carcinogenesis. Our results demonstrate that mobile element activation must be considered as one of the mechanisms when evaluating genomic damage/instability in response to environmental agents

TLE1 Is an Anoikis Regulator and Is Downregulated by Bit1 in Breast Cancer Cells.

TLE1 is a Groucho-related transcriptional repressor protein that exerts survival and antiapoptotic function in several cellular systems and has been implicated in the pathogenesis of cancer. In the present study, we found that TLE1 is a regulator of anoikis in normal mammary epithelial and breast carcinoma cells. The induction of apoptosis following loss of cell attachment to the extracellular matrix (anoikis) in untransformed mammary epithelial MCF10A cells was associated with significant downregulation of TLE1 expression. Forced expression of exogenous TLE1 in these cells promoted resistance to anoikis. In breast cancer cells, TLE1 expression was significantly upregulated following detachment from the extracellular matrix. Genetic manipulation of TLE1 expression via overexpression and downregulation approaches indicated that TLE1 promotes the anoikis resistance and anchorage-independent growth of breast carcinoma cells. Mechanistically, we show that TLE1 inhibits the Bit1 anoikis pathway by reducing the formation of the proapoptotic Bit1-AES complex in part through sequestration of AES in the nucleus. The mitochondrial release of Bit1 during anoikis as well as exogenous expression of the cytoplasmic localized Bit1 or its cell death domain induced cytoplasmic translocation and degradation of nuclear TLE1 protein. These findings indicate a novel role for TLE1 in the maintenance of anoikis resistance in breast cancer cells. This conclusion is supported by an immunohistochemical analysis of a breast cancer tissue array illustrating that TLE1 is selectively upregulated in invasive breast tumors relative to noninvasive ductal carcinoma in situ and normal mammary epithelial tissues

TLE1 Is an Anoikis Regulator and Is Downregulated by Bit1 in Breast Cancer Cells

TLE1 is a Groucho related transcriptional repressor protein that exerts survival and anti-apoptotic function in several cellular systems and has been implicated in the pathogenesis of cancer. In the present study, we found that TLE1 is a regulator of anoikis in normal mammary epithelial and breast carcinoma cells. The induction of apoptosis following loss of cell attachment to the extracellular matrix (anoikis) in untransformed mammary epithelial MCF10A cells was associated with significant downregulation of TLE1 expression. Forced expression of exogenous TLE1 in these cells promoted resistance to anoikis. In breast cancer cells, TLE1 expression was significantly upregulated following detachment from the ECM. Genetic manipulation of TLE1 expression via overexpression and downregulation approaches indicated that TLE1 promotes the anoikis resistance and anchorage-independent growth of breast carcinoma cells. Mechanistically, we show that TLE1 inhibits the Bit1 anoikis pathway by reducing the formation of the pro-apoptotic Bit1-AES complex in part through sequestration of AES in the nucleus. The mitochondrial release of Bit1 during anoikis as well as exogenous expression of the cytoplasmic localized Bit1 or its cell death domain (CDD) induced cytoplasmic translocation and degradation of nuclear TLE1 protein. These findings indicate a novel role for TLE1 in the maintenance of anoikis resistance in breast cancer cells. This conclusion is supported by an immunohistochemical analysis of a breast cancer tissue array illustrating that TLE1 is selectively upregulated in invasive breast tumors relative to noninvasive ductal carcinoma in situ (DCIS) and normal mammary epithelial tissues

Highly and Moderately Aggressive Mouse Ovarian cancer Cell Lines Exhibit Differential Gene Expression.

Patients with advanced epithelial ovarian cancer often experience disease recurrence after standard therapies, a critical factor in determining their five-year survival rate. Recent reports indicated that long-term or short-term survival is associated with varied gene expression of cancer cells. Thus, identification of novel prognostic biomarkers should be considered. Since the mouse genome is similar to the human genome, we explored potential prognostic biomarkers using two groups of mouse ovarian cancer cell lines (group 1: IG-10, IG-10pw, and IG-10pw/agar; group 2: IG-10 clones 2, 3, and 11) which display highly and moderately aggressive phenotypes in vivo. Mice injected with these cell lines have different survival time and rates, capacities of tumor, and ascites formations, reflecting different prognostic potentials. Using an Affymetrix Mouse Genome 430 2.0 Array, a total of 181 genes were differentially expressed (P \u3c 0.01) by at least twofold between two groups of the cell lines. Of the 181 genes, 109 and 72 genes were overexpressed in highly and moderately aggressive cell lines, respectively. Analysis of the 109 and 72 genes using Ingenuity Pathway Analysis (IPA) tool revealed two cancer-related gene networks. One was associated with the highly aggressive cell lines and affiliated with MYC gene, and another was associated with the moderately aggressive cell lines and affiliated with the androgen receptor (AR). Finally, the gene enrichment analysis indicated that the overexpressed 89 genes (out of 109 genes) in highly aggressive cell lines had a function annotation in the David database. The cancer-relevant significant gene ontology (GO) terms included Cell cycle, DNA metabolic process, and Programmed cell death. None of the genes from a set of the 72 genes overexpressed in the moderately aggressive cell lines had a function annotation in the David database. Our results suggested that the overexpressed MYC and 109 gene set represented highly aggressive ovarian cancer potential biomarkers while overexpressed AR and 72 gene set represented moderately aggressive ovarian cancer potential biomarkers. Based on our knowledge, the current study is first time to report the potential biomarkers relevant to different aggressive ovarian cancer. These potential biomarkers provide important information for investigating human ovarian cancer prognosis

Requirement of LaeA for Secondary Metabolism and Sclerotial Production in Aspergillus flavus

The nuclear regulator LaeA has been shown to govern production of multiple secondary metabolites in A. nidulans and A. fumigatus. Herein we examine the role of this protein in Aspergillus flavus. Similarly as in other Aspergilli, LaeA had a major effect on A. flavus secondary metabolism where ΔlaeA and over-expression laeA (OE::laeA) strains yielded opposite phenotypes resulting in decreased (increased) secondary metabolite production. The two mutant strains also exhibited striking morphological phenotypes in the loss (increase) of sclerotial production in comparison to wildtype. Growth on seed was marked by decreased (increased) conidial and aflatoxin production of the respective mutants; this was accompanied by decreased lipase activity in ΔlaeA, an enzymatic process correlated with seed maceration. Transcriptional examination of the mutants showed LaeA negatively regulates expression of its recently identified nuclear partner VeA, another global regulator of A. flavus secondary metabolites and sclerotia