Effects of partner proteins on BCA2 RING ligase activity

Abstract Background BCA2 is an E3 ligase linked with hormone responsive breast cancers. We have demonstrated previously that the RING E3 ligase BCA2 has autoubiquitination activity and is a very unstable protein. Previously, only Rab7, tetherin, ubiquitin and UBC9 were known to directly interact with BCA2. Methods Here, additional BCA2 binding proteins were found using yeast two-hybrid and bacterial-II-hybrid screening techniques with Human breast and HeLa cDNA libraries. Co-expression of these proteins was analyzed through IHC of TMAs. Investigation of the molecular interactions and effects were examined through a series of in vivo and in vitro assays. Results Ten unique BCA2 interacting proteins were identified, two of which were hHR23a and 14-3-3sigma. Both hHR23a and 14-3-3sigma are co-expressed with BCA2 in breast cancer cell lines and patient breast tumors (n = 105). hHR23a and BCA2 expression was significantly correlated (P = \u3c 0.0001 and P = 0.0113) in both nucleus and cytoplasm. BCA2 expression showed a statistically significant correlation with tumor grade. High cytoplasmic hHR23a trended towards negative nodal status. Binding to BCA2 by hHR23a and 14-3-3sigma was confirmed in vitro using tagged partner proteins and BCA2. hHR23a and 14-3-3sigma effect the autoubiquitination and auto-degradation activity of BCA2. Ubiquitination of hHR23a-bound BCA2 was found to be dramatically lower than that of free BCA2, suggesting that hHR23a promotes the stabilization of BCA2 by inactivating its autoubiquitination activity, without degradation of hHR23a. On the other hand, phosphorylated BCA2 protein is stabilized by interaction with 14-3-3sigma both with and without proteasome inhibitor MG-132 suggesting that BCA2 is regulated by multiple degradation pathways. Conclusions The interaction between BCA2 and hHR23a in breast cancer cells stabilizes BCA2. High expression of BCA2 is correlated with grade in breast cancer, suggesting regulation of this E3 ligase is important to cancer progression

Regulating BCA2: An Investigation into E3 Ligase Activity

The BCA2 E3 ligase is expressed in a majority of invasive breast cancers. BCA2 has inherent autoubiquitination activity which contributes to cell migration and proliferation processes. Here, ten novel BCA2 binding proteins were found using yeast and bacterial screening. Two of which were human homolog of Rad23 variant A (hHR23a) and 14-3-3σ. In vivo and in vitro assays confirmed that both hHR23a and 14-3-3σ bound BCA2 and were co-expressed with BCA2 in breast cancer cells. Interaction of BCA2 with hHR23a and 14-3-3σ affect the autoubiquitination and auto-degradation activity of BCA2. Multi-ubiquitination of hHR23a-bound BCA2 was dramatically lower than that of free BCA2, this corresponded to increased BCA2 expression and half-life. Furthermore, phosphorylated BCA2 protein was stabilized by interaction with 14-3-3σ, via substrate inhibition of BCA2 autoubiquitination. High expression of BCA2 is correlated with grade in breast cancer and regulation of this E3 ligase’s activity may be important to cancer progression.MAS

Loss of <i>Igfbp7</i> Causes Precocious Involution in Lactating Mouse Mammary Gland

<div><p>Background</p><p>Insulin like growth factors (IGFs) and their binding proteins (IGFBPs) are secreted peptides that play major roles in regulating the normal development and maturation of mammary gland. While <i>Igfbp7</i> has been shown to decrease breast tumor growth, its role in regulating the normal mammary gland development has not been studied. To this end, we generated <i>Igfbp7</i>-null mice and examined the development and maturation of mammary glands in the virgin, pregnant and lactating animals.</p><p>Results</p><p>We report here that loss of <i>Igfbp7</i> significantly retards mammary gland development in the virgin animals. More significantly, the pregnant <i>Igfpb7</i>-null glands contained fewer alveolar structures and that during lactation these glands exhibit the morphological changes that are associated with involution. The transcriptome profile of the <i>Igfbp7</i>-null glands on the lactation day 3 revealed a distinct involution-related gene signature compared to the lactating WT glands. Interestingly, we found that the lactating <i>Igfbp7</i>-null glands exhibit increased expression of <i>Stat3</i> and enhanced activation of (phosphorylated) <i>Stat3</i>, combined with decreased expression of <i>Stat5</i> suggesting that the absence of <i>Igfbp7</i> accelerates the onset of involution. We also found that in absence of <i>Igfpb7</i>, the lactating glands contain increased <i>Igfbp5</i> protein along with decreased expression of <i>IGF-1</i> Receptor and <i>Akt</i> activation. Finally, we show that during the normal course of involution, <i>Igfbp7</i> expression is significantly decreased in the mammary gland.</p><p>Conclusion</p><p>Our data suggest that loss of <i>Igfbp7</i> induces precocious involution possibly through diminished cell survival signals. Our findings identify <i>Igfbp7</i> as major regulator of involution in the mammary gland.</p></div

<i>Igfbp7</i> expression is substantially decreased during involution in the mammary gland.

<p>(A) To ascertain if expression of <i>Igfbp7</i> correlates with involution, protein extracts were prepared from the Wild Type glands on the lactation day 3 (WT LD3) or involuting WT glands (weaned for 5 days, WT Inv D5) and the expression of <i>Igfbp7</i> protein was examined using Western blots. A representative blot is shown and average <i>Igfbp7</i> expression was obtained from 3 independent protein extracts and depicted in the bar graph. As shown, <i>Igfbp7</i> expression is dramatically decreased during involution (*P<0.05). (B) <i>Igfbp7</i>^−/− transcript expression was examined using qPCR assays using RNA extracted from Wild Type 11 week-old virgin (WT 11Week), day 18.5 pregnant (WT D18.5 pregnant) or on lactation day 3 glands. The average transcript expressions obtained from 3 independent RNA samples are shown. While <i>Igfbp7</i> expression in the mammary gland does not show significant change during pregnancy, its expression is substantially increased during lactation. Statistical analysis was done through two-tailed t-test (***p<0.0005).</p

The <i>Igfbp7^−/−</i> transcriptome is enriched for involution-associate gene signature.

<p>(A) The transcriptome profiles of the Wild-Type (WT) and the <i>Igfbp7^−/−</i> glands on the lactation day 3 were analyzed using the RNA-Seq technique. Differentially expressed transcripts were identified by imposing a minimum of 1.5 fold change in the transcript expression level and a P-value of 0.05 or lower and are depicted on a Volcano plot. (B and C) The differentially expressed transcripts in the <i>Igfbp7^−/−</i> mammary epithelial cells (MECs) are categorized into the different KEGG Signaling Pathways using the DAVID Bioinformatics Resources. The fold enrichment scores are calculated based on the number of differentially regulated genes that belong to a particular KEGG signaling pathway out of the total gene set. The Benjamini statistics is used to identify the statistically significant gene enrichment for specific KEGG signaling pathways. Using this analysis the down regulated transcripts were found to be enriched in extracellular matrix receptor interaction, focal adhesion and cell adhesion molecules. The up regulated transcripts in the <i>Igfpb7</i>^−/− MECs showed enrichment for the WNT, TGF beta and adherens junction proteins. (D) To examine if the differentially regulated transcripts in the <i>Igfbp7</i>^−/− MECs show enrichment for involution-related genes, we first obtained a common involution gene signature by comparing 3 publically available transcriptome profile of involuting glands. Set 1 is from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087858#pone.0087858-Stein2" target="_blank">[35]</a>, Set 2 is from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087858#pone.0087858-Clarkson1" target="_blank">[34]</a>, Set 3 is from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087858#pone.0087858-Blanchard1" target="_blank">[33]</a>. The Ven diagram depicts a 72 gene-set that is common among all 3 data sets.</p

<i>Igfbp7^−/−</i> mice do not show expression of <i>Igfbp7</i> mRNA or protein.

<p>(A) Inguinal mammary glands from 11 week old virgin female mice were extracted from the wild-type mice (WT) or the knockout animals (<i>Igfbp7^−/−</i>) and where fixed and stained with an antibody specific to <i>Igfbp7</i> (green fluorescent color) and with propidium iodide (red color) to distinguish the nucleus of cells. As seen, <i>Igfbp7^−/−</i> glands do not show detectable expression of <i>Igfbp7</i> protein. (B) The <i>Igfbp7</i> transcript expression was measured using quantitative real time PCR. RNA was extracted from the 11-week-old inguinal WT or <i>Igfbp7^−/−</i> female mice and turned into cDNA. The transcript expression of <i>Igfbp7</i> was quantified relative to the <i>Gapdh</i> transcript levels. As can be seen the <i>Igfbp7</i> transcripts are at limit detection in the <i>Igfbp7^−/−</i> glands. Each data point is the average of 3 independent experiments.</p

<i>Igfbp7^−/−</i> glands show decreased proliferation index.

<p>(A) Inguinal mammary glands were isolated from wild-type (<i>Igfbp7^+/+</i>) or the <i>Igfbp7</i>^−/− mice on pregnancy days 9 (PD9) and 16 (PD16) as well as on lactation days 1 (LD1) and 3 (LD3) and at 3 weeks postpartum (3WPP). Sections from the formalin fixed and paraffin embedded glands were used to detect the expression of Ki-67 protein by Immunohistochemical stains. As can be seen, the <i>Igfbp7</i>^−/− glands show decreased number of cells positive for the expression of <i>Ki-67</i>. (B) The proliferation index of the WT and the <i>Igfbp7</i>^−/− glands are determined by examining the nuclear expression of Ki-67 protein in over 600 nuclei in the slide sections. The proliferation index is calculated by obtaining the percentage of <i>Ki-67</i> nuclei positive cells in each section. The average values obtained from 3 different fields and the standard deviations are plotted again the developmental stage of the mammary glands. P-values were obtained by performing two-tailed T-tests and provided in a table under the graph.</p

Loss of <i>Igfbp7</i> decreases the alveolar density of the mammary gland during the pregnancy and lactation.

<p>(A) Whole mount slides were prepared from inguinal glands extracted from the wild-type (+/+) or the <i>Igfbp7</i>-null (−/−) female mice during pregnancy days (PD) 9 and 16 and lactation days (LD) 1 and 3 as well as 3 weeks port partum (3WPP). The slides were photographed at 2x the magnification and a ruler was also photographed as a point of reference (the longer marks are in centimeters and the shorter marks are in millimeters). The insert pictures were obtained at 4x the magnification to allow more detailed analysis of the mammary structures. As demonstrated, loss of <i>Igfbp7</i> leads to marked decrease in the alveolar densities in the mammary glands at each of the developmental time points studied. (B) H&E stained sections were prepared from formalin fixed paraffin-embedded mammary gland on the PD6, PD16, LD1, LD3, and 3WPP. As can be seen, alveolar development is completely defective in the <i>Igfbp7^−/−</i> glands during pregnancy and lactation. The black arrows point to typical lobular structures in the WT or the <i>Igfbp7^−/−</i> glands. Compared to the WT, the <i>Igfbp7^−/−</i> glands contain smaller and partially closed alveolar sacks. Interestingly on the lactation day 3 the alveolar structures in the <i>Igfbp7^−/−</i> glands resemble the WT glands at 3WPP. (C) Western blot analysis showing decreased β-casein expression in the Igfbp7^−/− glands. Total protein was extracted from WT or Igfbp7^−/− glands on the lactation day 3, and size fractionated through western blots and the β-casein protein expression was determined and quantified relative to β-actin. One representative Western Blot is shown and the graph shows average of 2 independent protein extracts. As can be seen Igfbp7^−/− glands contain slightly less β-casein protein.</p