Ezrin mediates both HGF/Met autocrine and non-autocrine signaling-induced metastasis in melanoma.

Aberrant HGF/Met signaling promotes tumor migration, invasion, and metastasis through both autocrine and non-autocrine mechanisms; however, the molecular downstream signaling mechanisms by which HGF/Met induces metastasis are incompletely understood. We here report that Ezrin expression is stimulated by HGF and correlates with activated HGF/Met, indicating that HGF/Met signaling regulates the expression of Ezrin. We show that HGF/Met signaling activates the transcription factor Sp1 through the MAPK pathway, and activated Sp1 can in turn directly bind to the promoter of Ezrin gene and regulate its transcription. Notably, knockdown of Ezrin expression by shRNAs inhibits the metastasis induced by either HGF/Met autocrine or non-autocrine signaling in syngeneic wildtype and HGF transgenic mouse hosts. We also used small molecule drugs in preclinical mouse models to confirm that Ezrin is one of the downstream molecules mediating HGF/Met signaling-induced metastasis in melanoma. We conclude that Ezrin is a key downstream factor involved in the regulation of HGF/Met signaling-induced metastasis and demonstrate a link between Ezrin and HGF/Met/MAPK/Sp1 activation in the metastatic process. Our data indicate that Ezrin represents a promising therapeutic target for patients bearing tumors with activated HGF/Met signaling.

[BRCA1 selectively regulates the expression of progesterone receptor A in sporadic invasive ductal breast carcinoma].

OBJECTIVE: To investigate the expression level of BRCA1, progesterone receptor A (PRA) and B (PRB) in tissue of sporadic invasive ductal breast carcinoma and further statistically analyze the BRCA1 effects on the expression rates of PRA and PRB. METHODS: Sixty-eight cases of adenosis of breast and sporadic invasive ductal breast carcinoma were selected. The corresponding paraffin-embodied tissues were collected from the archive of Department of Pathology, Nanfang Hospital. The expressions level of BRCA1, PRA and PRB were detected by immunohistochemistry. Wilcoxon two-sample test was used to analyze the differential expression of BRCA1 between adenosis and sporadic invasive ductal breast carcinoma. Chi-square test was used to analyze the effects of BRCA1 on the PRA or PRB expression rate. P value < 0.05 was considered statistically significant. RESULTS: (1) In invasive sporadic ductal breast carcinoma, the positive rate of BRCA1 expression of 60.29% (41/68) was lower than the positive rate of BRCA1 expression at 85.30% (58/68) in adenosis of breast. And the difference of BRCA1 expression between two groups was statistically significant (P < 0.01); (2) In invasive sporadic ductal breast carcinoma, the positive rate of PRA expression for negative BRCA1 expression samples was 81.48% (22/27) and it was higher than the positive rate of PRA expression at 53.66% (22/47) for positive BRCA1 expression samples. And the difference of PRA expression rates between two groups was statistically significant (P < 0.05). It indicated that the expression of BRCA1 affected the expression rate of PRA; In invasive sporadic ductal breast carcinoma, the PRB expression rates between positive and negative BRCA1 expression samples were not statistically significant (P > 0.05). It indicated that BRCA1 had no effect upon the expression rate of PRB. CONCLUSION: In sporadic breast carcinoma, a negative expression of BRCA1 is selectively associated with a higher expression rate of PRA rather than PRB. Thus BRCA1 selectively regulates the expression of PRA in sporadic breast carcinoma.

Growth inhibition of human cancer cells by 5-aza-2'-deoxycytidine does not correlate with its effects on INK4a/ARF expression or initial promoter methylation status.

The cytotoxicity of 5-aza-2'-deoxycytidine (DAC) has been linked to demethylation of the INK4a/ARF tumor suppressor gene locus in various cell systems, but the causality of this association remains unproven. To test this assumption, we have examined the effects of DAC in two human cancer cell lines of differing INK4a/ARF promoter methylation status: MDA-MB-468 breast cancer cells in which INK4a/ARF is unmethylated and normally expressed, and DLD-1 colorectal cancer cells in which INK4a/ARF is methylated and repressed. In MDA-MB-468 cells, DAC induces cytotoxicity in the absence of any detectable increase of p14 or p16 expression, whereas small interfering RNA knockdown of p16/p14 expression fails to attenuate DAC cytotoxicity. In DLD-1 cells, DAC demethylates INK4a/ARF and restores both p16 and p14 expression at concentrations that fail to cause detectable growth inhibition or apoptosis; moreover, neither ARF nor INK4a transgene expression inhibits DLD-1 cell growth despite normalization of p14 and p16 expression. These data imply that neither of these cell lines depends on up-regulated expression of INK4a/ARF for DAC cytotoxicity. We propose that optimal anticancer use of this drug will await unambiguous identification of those DAC target genes primarily responsible for triggering growth inhibition, followed by clarification as to whether these upstream events are caused by hypomethylation or DNA damage.

[Blocking programmed death-ligand 1 attenuates maturation inhibition of dendritic cells by co-cultured breast cancer cells].

OBJECTIVE: To study if programmed death-ligand 1 (PL-L1) expression in breast cancer cell activates PD-L1/PD-1 pathway in dendritic cells to inhibit dendritic cell maturation. METHODS: Human monocytes were induced to differentiate into immature dendritic cells using GM-CSF and IL-4, and further to mature dendritic cells using TNF-α. PD-L1-expressing breast cancer cell line MDA-MB-231 was co-cultured in contact with the dendritic cells to observe the effects of the breast cancer cells on the maturation of the dendritic cells. A PD-L1 blocking antibody was applied to the co-culture, and the changes in the inhibitory effect of the MDA-MB-231 cells on dendritic cell maturation was observed. TNF-α-induced dendritic cells were treated with a recombinant human PD-L1 protein to study the effect of PD-L1/PD-1 pathway activation on the maturation of dendritic cells. The expression of PD-L1 in MDA-MB-231 cells and the dendritic cell maturation marker HLA-DR and CD83 were analyzed using flow cytometry. RESULTS: MDA-MB-231 cell line showed PD-L1 positivity on the cell membrane cells at a rate as high as (99.7∓0.15)%. In mature dendritic cells, the positivity rates for HLA-DR and CD83 were (88.8∓6.96)% and (18.36∓3.07)%, respectively, but in the co-culture system, the positivity rates of the dendritic cells were significantly decreased to (42.76∓10.52)% (P<0.01) and (9.93∓2.74)% (P<0.05), respectively, indicating that MDA-MB-231 cells inhibited the maturation of dendritic cells. Following treatment with a PD-L1 antibody isotype control, the percentages of HLA-DR- and CD83-positive cells in the co-culture were (45.17∓10.19)% and (10.15∓2.54)%, which were significantly increased to (63.46∓1.72)% and (16.46∓2.58)% after treatment with PD-L1 antibody, respectively (both P<0.05). Compared with the mature dendritic cell controls, the cells treated with the recombinant human PD-L1 protein exhibited significantly lowered percentages of HLA-DR-positive [from (84.23∓4.18)% to (2.56∓2.39)%, P<0.05] and CD83-positive cells [(87.26∓1.54)% to (60.67∓1.63)%, P<0.05]. CONCLUSION: The effect of PD-L1 antibody therapy on triple negative breast cancer can be partially mediated by blocking PD-L1 expression on breast cancer cell membrane, which attenuates the inhibition of dendritic cell maturation in the cancer microenvironment.

BRCA1 inhibition of telomerase activity in cultured cells.

Telomerase, an enzyme that maintains telomere length, plays major roles in cellular immortalization and cancer progression. We found that an exogenous BRCA1 gene strongly inhibited telomerase enzymatic activity in human prostate and breast cancer cell lines and caused telomere shortening in cell lines expressing wild-type BRCA1 (wtBRCA1) but not a tumor-associated mutant BRCA1 (T300G). wtBRCA1 inhibited the expression of the catalytic subunit (telomerase reverse transcriptase [TERT]) but had no effect on the expression of a subset of other components of the telomerase holoenzyme or on the expression of c-Myc, a transcriptional activator of TERT. However, endogenous BRCA1 associated and partially colocalized with c-Myc; exogenous wtBRCA1 strongly suppressed TERT promoter activity in various cell lines. The TERT inhibition was due, in part, to suppression of c-Myc E-box-mediated transcriptional activity. Suppression of TERT promoter and c-Myc activity required the amino terminus of BRCA1 but not the carboxyl terminus. Finally, endogenous BRCA1 and c-Myc were detected on transfected mouse and human TERT promoter segments in vivo. We postulate that inhibition of telomerase may contribute to the BRCA1 tumor suppressor activity.

Biogeography of cryoconite bacterial communities on glaciers of the Tibetan Plateau.

Cryoconite holes, water-filled pockets containing biological and mineralogical deposits that form on glacier surfaces, play important roles in glacier mass balance, glacial geochemistry and carbon cycling. The presence of cryoconite material decreases surface albedo and accelerates glacier mass loss, a problem of particular importance in the rapidly melting Tibetan Plateau. No studies have addressed the microbial community composition of cryoconite holes and their associated ecosystem processes on Tibetan glaciers. To further enhance our understanding of these glacial ecosystems on the Tibetan Plateau and to examine their role in carbon cycling as the glaciers respond to climate change, we explored the bacterial communities within cryoconite holes associated with three climatically distinct Tibetan Plateau glaciers using Illumina sequencing of the V4 region of the 16S rRNA gene. Cryoconite bacterial communities were dominated by Cyanobacteria, Chloroflexi, Betaproteobacteria, Bacteroidetes and Actinobacteria. Cryoconite bacterial community composition varied according to their geographical locations, exhibiting significant differences among glaciers studied. Regional beta diversity was driven by the interaction between geographic distance and environmental variables; the latter contributed more than geographic distance to the variation in cryoconite microbial communities. Our study is the first to describe the regional-scale spatial variability and to identify the factors that drive regional variability of cryoconite bacterial communities on the Tibetan Plateau.

[Nanog promotes the invasion of breast cancer cells by increasing PKCε expression].

OBJECTIVE: To study the relationship between Nanog-promoted metastasis of breast cancer and ezrin(T567) phosphorylation, and explore the possible mechanism by which Nanog regulates ezrin(T567) phosphorylation. METHODS: A siRNA construct targeting Nanog was transfected in breast cancer cells to knock down Nanog expression, and the changes in the cell invasion was detected using Transwell assay. The expression levels of Nanog and PKC and the phosphorylation level of ezrin(T567) were detected using Western blotting and immunofluorescent staining; the protein interaction between PKCε and ezrin was assayed by co-immunoprecipitation and Western blotting. RESULTS: Nanog knockdown significantly decreased the expression of PKCε protein, phosphorylation level of ezrin(T567) and the invasion ability of breast cancer cells. PKCε knockdown obviously decreased the phosphorylation level of ezrin(T567) in the cells, and PKCε and ezrin were co-immunoprecipitated. CONCLUDIONS: Nanogcan can upregulate the expression of PKCε to promote the phosphorylation of ezrin(T567), which can be a new mechanism by which Nanog promotes tumor metastasis.

[Phosphorylation of ezrin Tyr477 is critical in invasion of breast cancer cells stimulated by precursor of nerve growth factor].

OBJECTIVE: To investigate the effect of precursor of nerve growth factor (proNGF) in promoting invasion of breast cancer cells and its relation with ezrin expression and phosphorylation of ezrin Thr567 and Tyr477. METHODS: Human breast cancer cell lines MDA-MB-231 and MCF-7 were stimulated by gradient concentrations of proNGF (0, 2.5, 5 and 10 ng/mL) for 16 h, and the invasion of the cells was assessed with Transwell assay. The expression of ezrin and the phosphorylation of ezrin Thr567 and ezrin Tyr477 in the treated cells were examined by Western blotting. MDA-MB-231 cells were transfected with pEnter-His-ezrinY477F (a dominant negative mutant) to study the role of phosphrylation of ezrin Tyr477 in the invasion of breast cancer cell stimulated by proNGF. RESULTS: proNGF significantly promoted MDA-MB-231 and MCF-7 cell invasion in a concentration-dependent manner (P<0.05), and concentration- and time-dependently increased the phosphorylation of ezrin Tyr477 (P<0.05) without affecting the expression of ezrin or the phosphorylation of ezrin Thr567. The specific inhibitor of src, SKI-606, significantly inhibited the phosphorylation of ezrin Tyr477 induced by proNGF. Transfection with pEnter-His- ezrinY477F inhibited proNGF-induced invasion and phosphorylation of ezrin Tyr477 in MDA-MB-231 cells (P<0.05). CONCLUSION: Phosphorylation of ezrin Tyr477 plays a critical role in the invasion of breast cancer cells stimulated by proNGF via proNGF/src/ezrin Tyr477 pathway.

Role of BRCA1 in heat shock response.

The heat shock response is an evolutionarily conserved response to heat and other stresses that promotes the maintenance of key metabolic functions and cell survival. We report that exposure of human prostate (DU-145) and breast (MCF-7) cancer cells to heat (42 degrees C) caused a rapid disappearance of the breast cancer susceptibility gene-1 (BRCA1) protein, starting at approximately 1 h after the onset of heating and slightly lagging behind the increase in heat shock protein 70 (HSP70) levels. The heat-induced loss of BRCA1 occurred at the protein level, since: (1) BRCA1 mRNA expression was unaffected; and (2) the BRCA1 protein loss was also observed in DU-145 cells that expressed exogenous wild-type BRCA1 (wtBRCA1). In addition to heat regulation of BRCA1 protein levels, we also found that BRCA1 could modulate the heat shock response. Thus, wtBRCA1 overexpressing DU-145 cell clones showed significantly decreased sensitivity to heat-induced cytotoxicity; and Brca1 mutant mouse embryo fibroblasts showed increased sensitivity to heat. The DU-145 wtBRCA1 clones also showed increased expression of the small heat shock protein HSP27; and reporter assays revealed that wtBRCA1 stimulated a two to four-fold increase in HSP27 promoter activity, consistent with its ability to upregulate HSP27 mRNA and protein levels. In studies using epitope-tagged truncated BRCA1 proteins, the ability to stimulate the HSP27 promoter and to mediate heat-induced degradation required the amino-terminus but not the carboxyl-terminus of BRCA1. Although the heat-induced loss of BRCA1 appeared to be due to protein degradation, various protein metabolic agents (or combinations) failed to block this event, including: MG132 (a 26S proteasomal inhibitor), N-acetyl-leucyl-leucyl-norleucinal (a calpain inhibitor), z-VAD-fmk (a pan-caspase inhibitor), and ammonium chloride and chloroquine (which stabilize lysosomes). These findings suggest that in addition to its other functions, BRCA1 may participate in mammalian heat shock response pathways.

Functional potential of soil microbial communities in the maize rhizosphere.

Microbial communities in the rhizosphere make significant contributions to crop health and nutrient cycling. However, their ability to perform important biogeochemical processes remains uncharacterized. Here, we identified important functional genes that characterize the rhizosphere microbial community to understand metabolic capabilities in the maize rhizosphere using the GeoChip-based functional gene array method. Significant differences in functional gene structure were apparent between rhizosphere and bulk soil microbial communities. Approximately half of the detected gene families were significantly (p<0.05) increased in the rhizosphere. Based on the detected gyrB genes, Gammaproteobacteria, Betaproteobacteria, Firmicutes, Bacteroidetes and Cyanobacteria were most enriched in the rhizosphere compared to those in the bulk soil. The rhizosphere niche also supported greater functional diversity in catabolic pathways. The maize rhizosphere had significantly enriched genes involved in carbon fixation and degradation (especially for hemicelluloses, aromatics and lignin), nitrogen fixation, ammonification, denitrification, polyphosphate biosynthesis and degradation, sulfur reduction and oxidation. This research demonstrates that the maize rhizosphere is a hotspot of genes, mostly originating from dominant soil microbial groups such as Proteobacteria, providing functional capacity for the transformation of labile and recalcitrant organic C, N, P and S compounds.

[Tricostantin A inhibits self-renewal of breast cancer stem cells in vitro].

OBJECTIVE: To investigate the effect of tricostantin A (TSA) on self-renewal of breast cancer stem cells and explore the mechanisms. METHODS: Breast cancer cell lines MDA-MB-468, MDA-MB-231, MCF-7 and SKBR3 were cultured in suspension and treated with different concentrations of TSA for 7 days, using 0.1% DMSO as the control. Secondary mammosphere formation efficiency and percentage of CD44(+)/CD24(-) sub-population in the primary mammospheres were used to evaluate the effects of TSA on self-renewal of breast cancer stem cells. The breast cancer stem cell surface marker CD44(+)/CD24(-) and the percentage of apoptosis in the primary mammospheres were assayed using flow cytometry. The mRNA expressions of Nanog, Sox2 and Oct4 in the primary mammospheres were assayed with quantitative PCR. RESULTS: TSA at both 100 and 500 nmol/L, but not at 10 nmol/L, partially inhibited the self-renewal of breast cancer stem cells from the 4 cell lines. TSA at 500 nmol/L induced cell apoptosis in the primary mammospheres. TSA down-regulated the mRNA expression of Nanog and Sox2 in the primary mammospheres. CONCLUSION: TSA can partially inhibit the self-renewal of breast cancer stem cells through a mechanism involving the down-regulation of Nanog and Sox2 expression, indicating the value of combined treatments with low-dose TSA and other anticancer drugs to achieve maximum inhibition of breast cancer stem cell self-renewal. The core transcriptional factor of embryonic stem cells Nanog and Sox2 can be potential targets of anticancer therapy.

[BRCA1 inhibits progesterone-induced proliferation and migration of breast cancer cells].

OBJECTIVE: To study the effect of BRCA1 in regulating the proliferation and migration of breast cancer cells stimulated by progesterone. METHODS: Breast cancer MCF-7 and T-47D cell were transfected with a vector containing the coding sequence of BRCA1 (pFlag-CMV2-BRCA1 wt) to induce BRCA1 overexpression or with the empty vector (control). The cells were then stimulated with progesterone, and the cell proliferation and migration were observed using MTT assay and wound healing assay, respectively. The proliferation and migration of MCF-7 cells were also observed following transfection with a small interfering RNA (siRNA) for BRCA1 knockdown or with a scrambled siRNA prior to progesterone stimulation. RESULTS: Transfection with the empty vector and with pFlag-CMV2-BRCA1 wt prior to progesterone stimulation caused significantly different proliferation rates in MCF-7 cells [(114.4∓6.0)% vs (82.1∓3.2)%, P<0.05] and in T-47D cells [(111.3∓4.3)% vs (84.2∓3.5)%, P<0.05], resulting also in significantly different cell migration rates (55.9% vs 15.8% in MCF-7 cells and 44.83% vs 10.43% in T-47D cells). Compared to the scrambled siRNA, BRCA1 siRNA transfection prior to progesterone stimulation significantly increased the proliferation rates [(114.4∓3.05)% vs (125.3∓4.0)%, P<0.05] and migration rate (39.2% vs 69.08%) of MCF-7 cells. The progesterone antagonist RU468 could antagonize the effects of BRCA1 knockdown in enhancing progesterone-stimulated MCF-7 cell proliferation and migration. CONCLUSION: A decreased BRCA1 expression can enhance progesterone-stimulated tumor cell proliferation and migration in sporadic breast cancer.

Epigenetic drugs can stimulate metastasis through enhanced expression of the pro-metastatic Ezrin gene.

Ezrin has been reported to be upregulated in many tumors and to participate in metastatic progression. No study has addressed epigenetic modification in the regulation of Ezrin gene expression, the importance of which is unknown. Here, we report that highly metastatic rhabdomyosarcoma (RMS) cells with high levels of Ezrin have elevated acetyl-H3-K9 and tri-methyl-H3-K4 as well as reduced DNA methylation at the Ezrin gene promoter. Conversely, poorly metastatic RMS cells with low levels of Ezrin have reduced acetyl-H3-K9 and elevated methylation. Thus epigenetic covalent modifications to histones within nucleosomes of the Ezrin gene promoter are linked to Ezrin expression, which in fact can be regulated by epigenetic mechanisms. Notably, treatment with histone deacetylase (HDAC) inhibitors or DNA demethylating agents could restore Ezrin expression and stimulate the metastatic potential of poorly metastatic RMS cells characterized by low Ezrin levels. However, the ability of epigenetic drugs to stimulate metastasis in RMS cells was inhibited by expression of an Ezrin-specific shRNA. Our data demonstrate the potential risk associated with clinical application of broadly acting covalent epigenetic modifiers, and highlight the value of combination therapies that include agents specifically targeting potent pro-metastatic genes.

[Progesterone promotes the proliferation and migration of cultured breast cancer cells].

OBJECTIVE: To investigate the effects of progesterone on the growth and migration of breast cancer cells. METHODS: MCF-7 and T-47D cells were cultured in DMEM and stimulated with 100 nmol/L progesterone for 48 h, and the cell proliferation was evaluated by MTT assay, cell migration by wound-healing assay and E-catherin expression by Western blotting. RESULTS: Progesterone stimulated the cell proliferation and migration and down-regulated the expression of E-catherin in both MCF-7 and T-47D cells. CONCLUSIONS: Progesterone stimulates the cell proliferation and migration of cultured breast cancer cells, suggesting the clinical significance of anti-progesterone therapy in breast cancer.

[BRCA1 regulates progesterone receptors A and B protein expressions in breast cancer cells in vitro].

OBJECTIVE: To study the regulatory role of BRCA1 in the expression of progesterone receptors A and B (PRA and PRB) in breast cancer cells. METHODS: Breast cancer MCF-7 cells were transfected with pFlag-CMV2-BRCA1 wt plasmid containing a full-length BRCA1 cDNA or with BRCA1-specific siRNA via lipofectamine 2000 to induce overexpression or suppressed expression of BRCA1, respectively. Twenty-four hours after the transfection, the cells were incubated in fresh culture medium containing 100 nmol/L progesterone for 24 h. The total RNA extract or whole cell lysate was prepared for detecting BRCA1, PRA and PRB expressions using RT-PCR and Western blotting. RESULTS: The protein expressions of PRA and PRB were significantly decreased whereas their mRNA expressions remained unchanged in MCF-7 cells overexpressing BRCA1. In MCF-7 cells with BRCA1 knock-down, in contrast, the PRA and PRB protein expressions were markedly increased. CONCLUSION: In breast cancer cells, exogenous and endogenous BRCA1 can both down-regulate the expressions of PRA and PRB at the protein level.