TP53 Status as a Determinant of Pro- vs Anti-Tumorigenic Effects of Estrogen Receptor-Beta in Breast Cancer.

BACKGROUND: Anti-tumorigenic vs pro-tumorigenic roles of estrogen receptor-beta (ESR2) in breast cancer remain unsettled. We investigated the potential of TP53 status to be a determinant of the bi-faceted role of ESR2 and associated therapeutic implications for triple negative breast cancer (TNBC). METHODS: ESR2-TP53 interaction was analyzed with multiple assays including the in situ proximity ligation assay. Transcriptional effects on TP53-target genes and cell proliferation in response to knocking down or overexpressing ESR2 were determined. Patient survival according to ESR2 expression levels and TP53 mutation status was analyzed in the basal-like TNBC subgroup in the Molecular Taxonomy of Breast Cancer International Consortium (n = 308) and Roswell Park Comprehensive Cancer Center (n = 46) patient cohorts by univariate Cox regression and log-rank test. All statistical tests are two-sided. RESULTS: ESR2 interaction with wild-type and mutant TP53 caused pro-proliferative and anti-proliferative effects, respectively. Depleting ESR2 in cells expressing wild-type TP53 resulted in increased expression of TP53-target genes CDKN1A (control group mean [SD] = 1 [0.13] vs ESR2 depletion group mean [SD] = 2.08 [0.24], P = .003) and BBC3 (control group mean [SD] = 1 [0.06] vs ESR2 depleted group mean [SD] = 1.92 [0.25], P = .003); however, expression of CDKN1A (control group mean [SD] = 1 [0.21] vs ESR2 depleted group mean [SD] = 0.56 [0.12], P = .02) and BBC3 (control group mean [SD] = 1 [0.03] vs ESR2 depleted group mean [SD] = 0.55 [0.09], P = .008) was decreased in cells expressing mutant TP53. Overexpressing ESR2 had opposite effects. Tamoxifen increased ESR2-mutant TP53 interaction, leading to reactivation of TP73 and apoptosis. High levels of ESR2 expression in mutant TP53-expressing basal-like tumors is associated with better prognosis (Molecular Taxonomy of Breast Cancer International Consortium cohort: log-rank P = .001; hazard ratio = 0.26, 95% confidence interval = 0.08 to 0.84, univariate Cox P = .02). CONCLUSIONS: TP53 status is a determinant of the functional duality of ESR2. Our study suggests that ESR2-mutant TP53 combination prognosticates survival in TNBC revealing a novel strategy to stratify TNBC for therapeutic intervention potentially by repurposing tamoxifen.

Dataset of STAT5A status in breast cancer.

We analysed STAT5A gene expression in breast cancer using the Oncomine database. We exemplify four representative studies showing that STAT5A is generally downregulated in breast cancer.

STAT5A is regulated by DNA damage via the tumor suppressor p53.

Here we report that the STAT5A transcription factor is a direct p53 transcriptional target gene. STAT5A is well expressed in p53 wild type cells but not in p53-null cells. Inhibition of p53 reduces STAT5A expression. DNA damaging agents such as doxorubicin also induced STAT5A expression in a p53 dependent manner. Two p53 binding sites were mapped in the STAT5A gene and named PBS1 and PBS2; these sites were sufficient to confer p53 responsiveness in a luciferase reporter gene. Chromatin immunoprecipitation experiments revealed that PBS2 has constitutive p53 bound to it, while p53 binding to PBS1 required DNA damage. In normal human breast lobules, weak p53 staining correlated with regions of intense STAT5A staining. Interestingly, in a cohort of triple negative breast tumor tissues there was little correlation between regions of p53 and STAT5A staining, likely reflecting a high frequency of p53 mutations that stabilize the protein in these tumors. We thus reveal an unexpected connection between cytokine signaling and p53.

Cdc42-interacting protein 4 is a Src substrate that regulates invadopodia and invasiveness of breast tumors by promoting MT1-MMP endocytosis.

Invadopodia are actin-rich membrane protrusions that promote extracellular matrix degradation and invasiveness of tumor cells. Src protein-tyrosine kinase is a potent inducer of invadopodia and tumor metastases. Cdc42-interacting protein 4 (CIP4) adaptor protein interacts with actin regulatory proteins and regulates endocytosis. Here, we show that CIP4 is a Src substrate that localizes to invadopodia in MDA-MB-231 breast tumor cells expressing activated Src (MDA-SrcYF). To probe the function of CIP4 in invadopodia, we established stable CIP4 knockdown in MDA-SrcYF cell lines by RNA interference. Compared with control cells, CIP4 knockdown cells degrade more extracellular matrix (ECM), have increased numbers of mature invadopodia and are more invasive through matrigel. Similar results are observed with knockdown of CIP4 in EGF-treated MDA-MB-231 cells. This inhibitory role of CIP4 is explained by our finding that CIP4 limits surface expression of transmembrane type I matrix metalloprotease (MT1-MMP), by promoting MT1-MMP internalization. Ectopic expression of CIP4 reduces ECM digestion by MDA-SrcYF cells, and this activity is enhanced by mutation of the major Src phosphorylation site in CIP4 (Y471). Overall, our results identify CIP4 as a suppressor of Src-induced invadopodia and invasion in breast tumor cells by promoting endocytosis of MT1-MMP.

Doubles game: Src-Stat3 versus p53-PTEN in cellular migration and invasion.

We have recently shown that Src induces the formation of podosomes and cell invasion by suppressing endogenous p53, while enhanced p53 strongly represses the Src-induced invasive phenotype. However, the mechanism by which Src and p53 play antagonistic roles in cell invasion is unknown. Here we show that the Stat3 oncogene is a required downstream effector of Src in inducing podosome structures and related invasive phenotypes. Stat3 promotes Src phenotypes through the suppression of p53 and the p53-inducible protein caldesmon, a known podosome antagonist. In contrast, enhanced p53 attenuates Stat3 function and Src-induced podosome formation by upregulating the tumor suppressor PTEN. PTEN, through the inactivation of Src/Stat3 function, also stabilizes the podosome-antagonizing p53/caldesmon axis, thereby further enhancing the anti-invasive potential of the cell. Furthermore, the protein phosphatase activity of PTEN plays a major role in the negative regulation of the Src/Stat3 pathway and represses podosome formation. Our data suggest that cellular invasiveness is dependent on the balance between two opposing forces: the proinvasive oncogenes Src-Stat3 and the anti-invasive tumor suppressors p53-PTEN.

p53 suppresses Src-induced podosome and rosette formation and cellular invasiveness through the upregulation of caldesmon.

The tumor-suppressive role of p53 at the level of tumor initiation is well documented. It has also been shown previously that p53 acts against tumor progression/metastasis. However, its role in modulating cell migration and invasion leading to metastasis is poorly understood. In this study, using vascular smooth muscle cells and NIH 3T3 fibroblast cells, we have shown that p53 potently suppresses Src-induced podosome/rosette formation, extracellular matrix digestion, cell migration, and invasion. The overexpression of exogenous wild-type p53 or the activation of the endogenous p53 function suppresses, while the short hairpin RNA-mediated knockdown of p53 expression or the pageing of its function exacerbates, Src-induced migratory and invasive phenotypes. We have also found that p53 expression and function are downregulated in cells stably transformed with constitutively active Src that exhibit aggressive invasive properties. Lastly, p53 upregulates the expression of caldesmon, an actin-binding protein that has been shown to be an inhibitor of podosome/invadopodium formation. The ability of p53 to suppress Src phenotypes in transformed cells was largely abolished by knocking down caldesmon. This study reports a novel molecular mechanism (caldesmon), as well as a structural basis (podosomes/rosettes), to show how p53 can act as an anti-motility/invasion/metastasis agent.

Novel multiplex PCR approaches for the simultaneous detection of human pathogens: Escherichia coli 0157:H7 and Listeria monocytogenes.

Escherichia coli 0157:H7 and Listeria monocytogenes are the two most important food-borne human pathogens. To develop a single, rapid and sensitive PCR based test for simultaneous detection of both the organisms, fliCh7 and iap gene specific primers were used respectively for E. coli 0157:H7 and L. monocytogenes. Initially, with equal quantities of purified genomic DNAs of these organisms a multiplex PCR reaction was standardized to yield uniform amplification of both targets. Although, this assay detected E. coli 0157:H7 with high sensitivity, it failed to pick up L. monocytogenes after several hours of enrichment in broth medium initially spiked with equal numbers of live cells. This was found to be due to unequal growth of these organisms leading to disparity in the amount of template DNAs represented in the DNA preparation applied for conventional multiplex PCR amplification. To circumvent this, we have developed a modified method of enrichment and harvesting leading to highly sensitive and rapid single reaction PCR detection of both pathogens. We have also successfully developed two novel multiplex PCR formats for the generation of uniform PCR signals. Some of these methods might find broader application for the simultaneous detection of different combinations of multiple pathogens.

An E2F/miR-20a autoregulatory feedback loop.

The E2F family of transcription factors is essential in the regulation of the cell cycle and apoptosis. While the activity of E2F1-3 is tightly controlled by the retinoblastoma family of proteins, the expression of these factors is also regulated at the level of transcription, post-translational modifications and protein stability. Recently, a new level of regulation of E2Fs has been identified, where micro-RNAs (miRNAs) from the mir-17-92 cluster influence the translation of the E2F1 mRNA. We now report that miR-20a, a member of the mir-17-92 cluster, modulates the translation of the E2F2 and E2F3 mRNAs via binding sites in their 3'-untranslated region. We also found that the endogenous E2F1, E2F2, and E2F3 directly bind the promoter of the mir-17-92 cluster activating its transcription, suggesting an autoregulatory feedback loop between E2F factors and miRNAs from the mir-17-92 cluster. Our data also point toward an anti-apoptotic role for miR-20a, since overexpression of this miRNA decreased apoptosis in a prostate cancer cell line, while inhibition of miR-20a by an antisense oligonucleotide resulted in increased cell death after doxorubicin treatment. This anti-apoptotic role of miR-20a may explain some of the oncogenic capacities of the mir-17-92 cluster. Altogether, these results suggest that the autoregulation between E2F1-3 and miR-20a is important for preventing an abnormal accumulation of E2F1-3 and may play a role in the regulation of cellular proliferation and apoptosis.

DNA damage signaling and p53-dependent senescence after prolonged beta-interferon stimulation.

Interferons are cytokines with potent antiviral and antiproliferative activities. We report that although a transient exposure to beta-interferon induces a reversible cell cycle arrest, a sustained treatment triggers a p53-dependent senescence program. Beta-interferon switched on p53 in two steps. First, it induced the acetylation of p53 at lysine 320 and its dephosphorylation at serine 392 but not p53 activity. Later on, it triggered a DNA signaling pathway, the phosphorylation of p53 at serine 15 and its transcriptional activity. In agreement, beta-interferon-treated cells accumulated gamma-H2AX foci and phosphorylated forms of ATM and CHK2. The DNA damage signaling pathway was activated by an increase in reactive oxygen species (ROS) induced by interferon and was inhibited by the antioxidant N-acetyl cysteine. More important, RNA interference against ATM inhibited p53 phosphorylation at serine 15, p53 activity and senescence in response to beta-interferon. Beta-interferon-induced senescence was more efficient in cells expressing either, p53, or constitutive allele of ERK2 or RasV12. Hence, beta-interferon-induced senescence targets preferentially cells with premalignant changes.

RNA silencing of checkpoint regulators sensitizes p53-defective prostate cancer cells to chemotherapy while sparing normal cells.

p53 is frequently mutated in patients with prostate cancer, especially in those with advanced disease. Therefore, the selective elimination of p53 mutant cells will likely have an impact in the treatment of prostate cancer. Because p53 has important roles in cell cycle checkpoints, it has been anticipated that modulation of checkpoint pathways should sensitize p53-defective cells to chemotherapy while sparing normal cells. To test this idea, we knocked down ataxia telangiectasia mutated (ATM) gene by RNA interference in prostate cancer cell lines and in normal human diploid fibroblasts IMR90. ATM knockdown in p53-defective PC3 prostate cancer cells accelerated their cell cycle transition, increased both E2F activity and proliferating cell nuclear antigen expression, and compromised cell cycle checkpoints, which are normally induced by DNA damage. Consequently, PC3 cells were sensitized to the killing effects of the DNA-damaging drug doxorubicin. Combining ATM knockdown with the Chk1 inhibitor UCN-01 further increased doxorubicin sensitivity in these cells. In contrast, the same strategy did not sensitize either IMR90 or LNCaP prostate cancer cells, both of which have normal p53. However, IMR90 and LNCaP cells became more sensitive to doxorubicin or doxorubicin plus UCN-01 when both p53 and ATM functions were suppressed. In addition, knockdown of the G(2) checkpoint regulators ATR and Chk1 also sensitized PC3 cells to doxorubicin and increased the expression of the E2F target gene PCNA. Together, our data support the concept of selective elimination of p53 mutant cells by combining DNA damage with checkpoint inhibitors and suggest a novel mechanistic insight into how such treatment may selectively kill tumor cells.

Isolation, purification and characterization of chymosin from riverine buffalo (Bubalos bubalis).

Chymosin, an aspartyl proteinase, is used for curdling of milk and manufacture of cheese. We report the purification and the physicochemical properties of chymosin isolated from the abomasal tissue of buffalo calves. The enzyme preparation extracted from buffalo abomasal tissues could be purified 29-fold using anion exchange and gel filtration chromatography. The molecular weight of the purified enzyme was 35.6 kDa on SDS-PAGE. Partial N-terminal amino acid sequence of the first eight amino acid sequences of buffalo chymosin was identical to the first eight amino acid sequences of cattle chymosin. Buffalo chymosin exhibited a skewed bell-shaped stability profile as a function of temperature with maximum activity near 55 degrees C. Milk clotting activity decreased gradually as pH increased. The enzyme became completely inactive, however, above pH 7.0. The ratio of milk clotting to proteolytic activity was 3.03. When compared with cattle chymosin, there were subtle differences in the stability and relative proteolytic activity of buffalo chymosin.