Eukaryotic initiation factor 4E-binding protein as an oncogene in breast cancer

Abstract Background Eukaryotic Initiation Factor 4E-Binding Protein (EIF4EBP1, 4EBP1) is overexpressed in many human cancers including breast cancer, yet the role of 4EBP1 in breast cancer remains understudied. Despite the known role of 4EBP1 as a negative regulator of cap-dependent protein translation, 4EBP1 is predicted to be an essential driving oncogene in many cancer cell lines in vitro, and can act as a driver of cancer cell proliferation. EIF4EBP1 is located within the 8p11-p12 genomic locus, which is frequently amplified in breast cancer and is known to predict poor prognosis and resistance to endocrine therapy. Methods Here we evaluated the effect of 4EBP1 targeting using shRNA knock-down of expression of 4EBP1, as well as response to the mTORC targeted drug everolimus in cell lines representing different breast cancer subtypes, including breast cancer cells with the 8p11-p12 amplicon, to better define a context and mechanism for oncogenic 4EBP1. Results Using a genome-scale shRNA screen on the SUM panel of breast cancer cell lines, we found 4EBP1 to be a strong hit in the 8p11 amplified SUM-44 cells, which have amplification and overexpression of 4EBP1. We then found that knock-down of 4EBP1 resulted in dramatic reductions in cell proliferation in 8p11 amplified breast cancer cells as well as in other luminal breast cancer cell lines, but had little or no effect on the proliferation of immortalized but non-tumorigenic human mammary epithelial cells. Kaplan-Meier analysis of EIF4EBP1 expression in breast cancer patients demonstrated that overexpression of this gene was associated with reduced relapse free patient survival across all breast tumor subtypes. Conclusions These results are consistent with an oncogenic role of 4EBP1 in luminal breast cancer and suggests a role for this protein in cell proliferation distinct from its more well-known role as a regulator of cap-dependent translation.https://deepblue.lib.umich.edu/bitstream/2027.42/149184/1/12885_2019_Article_5667.pd

CTD2 Database 2017 for EIF4EBP1

CTD2 Dashboard compiled experimental observations across network findings as of November 2017 for EIF4EBP

Relative contributions of norspermidine synthesis and signaling pathways to the regulation of Vibrio cholerae biofilm formation.

The polyamine norspermidine is one of the major polyamines synthesized by Vibrionales and has also been found in various aquatic organisms. Norspermidine is among the environmental signals that positively regulate Vibrio cholerae biofilm formation. The NspS/MbaA signaling complex detects extracellular norspermidine and mediates the response to this polyamine. Norspermidine binding to the NspS periplasmic binding protein is thought to inhibit the phosphodiesterase activity of MbaA, increasing levels of the biofilm-promoting second messenger cyclic diguanylate monophosphate, thus enhancing biofilm formation. V. cholerae can also synthesize norspermidine using the enzyme NspC as well as import it from the environment. Deletion of the nspC gene was shown to reduce accumulation of bacteria in biofilms, leading to the conclusion that intracellular norspermidine is also a positive regulator of biofilm formation. Because V. cholerae uses norspermidine to synthesize the siderophore vibriobactin it is possible that intracellular norspermidine is required to obtain sufficient amounts of iron, which is also necessary for robust biofilm formation. The objective of this study was to assess the relative contributions of intracellular and extracellular norspermidine to the regulation of biofilm formation in V. cholerae. We show the biofilm defect of norspermidine synthesis mutants does not result from an inability to produce vibriobactin as vibriobactin synthesis mutants do not have diminished biofilm forming abilities. Furthermore, our work shows that extracellular, but not intracellular norspermidine, is mainly responsible for promoting biofilm formation. We establish that the NspS/MbaA signaling complex is the dominant mediator of biofilm formation in response to extracellular norspermidine, rather than norspermidine synthesized by NspC or imported into the cell

Role of NspS, MbaA, and NspC on biofilm formation in <i>V</i>. <i>cholerae</i>.

<p><b>(A)</b> Biofilm assay of <i>ΔnspS</i>, <i>nspC</i>::<i>kan</i>^<i>R</i>, and <i>nspC</i>::<i>kan</i>^<i>R</i><i>ΔnspS</i> mutations, with and without exogenous norspermidine. <b>(B)</b> Biofilm assay of <i>nspC</i>::<i>kan</i>^<i>R</i>, <i>ΔmbaA</i>, and <i>nspC</i>::<i>kan</i>^<i>R</i><i>ΔmbaA</i> mutations, with and without exogenous norspermidine. Biofilms were formed in borosilicate tubes in LB broth for 18 h at 27°C and quantified as described in Materials and Methods. Error bars show standard deviations of three biological replicates. A star indicates a statistically significant difference between wild type and the mutants. A double star indicates a statistically significant difference between growth media conditions. A p-value <0.05 was considered significant. WT, wild type.</p

Effects of vibriobactin synthesis and utilization on biofilm formation in <i>V</i>. <i>cholerae</i>.

<p><b>(A)</b> Biofilm formation of <i>ΔvibF</i> and <i>viuA</i>::<i>tet</i>^<i>R</i> mutants. <b>(B)</b> Biofilm formation of <i>ΔvibF</i>, <i>nspC</i>::<i>kan</i>^<i>R</i>, and <i>nspC</i>::<i>kan</i>^<i>R</i><i>ΔvibF</i> mutants. Biofilms were formed in borosilicate tubes in LB broth for 24 h at 27°C and quantified as described in Materials and Methods. EDDA was added to chelate iron to generate iron-deplete conditions. Error bars show standard deviations of three biological replicates. A star indicates a statistically significant difference between wild type and the mutants. A double star indicates a statistically significant difference between growth media conditions. A p-value <0.05 was considered significant. WT, wild type.</p

Effects of <i>ΔpotD1</i>, <i>nspC</i>::<i>kan</i>^<i>R</i>, <i>nspC</i>::<i>kan</i>^<i>R</i><i>ΔpotD1</i>, and <i>nspC</i>::<i>kan</i>^<i>R</i><i>ΔnspSΔpotD1</i> mutations, with and without exogenous norspermidine, on biofilm formation in <i>V</i>. <i>cholerae</i>.

<p>Biofilms were formed in borosilicate tubes in LB broth for 18 h at 27°C and quantified as described in Materials and Methods. Error bars show standard deviations of three biological replicates. A star indicates a statistically significant difference between wild type and the mutants. A double star indicates a statistically significant difference between growth media conditions. A p-value <0.05 was considered significant. WT, wild type. The values for WT and <i>nspC</i>::<i>kan</i>^<i>R</i> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186291#pone.0186291.g004" target="_blank">Fig 4</a> are the same as these experiments were performed simultaneously.</p

Role of NspS and NspC on cellular polyamine content in <i>V</i>. <i>cholerae</i>. Polyamine composition of <i>nspC</i>::<i>kan</i>^<i>R</i><i>ΔnspS</i> cells with and without exogenous norspermidine.

<p>Polyamines were extracted from cells, derivatized by benzoylation and analyzed by HPLC as described in Materials and Methods. Labeled peaks on the chromatogram correspond to putrescine (put), diaminopropane (dap), cadaverine (cad), norspermidine (nspd), and spermidine (spd). AU₂₅₄, absorbance units at 254 nm. Only 4–14 minutes of a 40-minute run are plotted for clarity.</p

Artificially increasing c-di-GMP levels can overcome the <i>nspS</i> defect.

<p><b>(A)</b> Biofilm assay of <i>ΔnspS</i> with <i>qrgB</i> and <i>ΔnspS</i> with <i>qrgB</i>_AADEF mutants. <b>(B)</b> Biofilm assay of <i>nspC</i>::<i>kan</i>^<i>R</i><i>ΔpotD1ΔnspS</i> with <i>qrgB</i> and <i>nspC</i>::<i>kan</i>^<i>R</i><i>ΔpotD1ΔnspS</i> with <i>qrgB</i>_AADEF mutants. Biofilms were formed in borosilicate tubes in LB broth for 18 h at 37°C and quantified as described in Materials and Methods. Relative biomass was calculated using the following equation OD₆₅₅ mutant/OD₆₅₅ wild type (Y-axis). Error bars show standard deviations of three biological replicates. A star indicates a statistically significant difference between wild type and the mutants. A p-value <0.05 was considered significant. WT, wild type.</p

Proposed environmental model.

<p>Environmental norspermidine may primarily derive from endogenously-produced norspermidine that is released during cell lysis or exported to the periplasm by an unknown transporter. It may also be provided by nearby eukaryotic organisms. Norspermidine may also act as a quorum sensing molecule, allowing <i>V</i>. <i>cholerae</i> to detect this signal, recognize that it is in the presence of other <i>Vibrios</i>, and respond appropriately by forming the <i>Vibrio</i> polysaccharide. In this way, norspermidine may allow <i>V</i>. <i>cholerae</i> to persist in its biofilm form in its natural environment.</p

Primers.

<p>Primers.</p