Regulation of CNKSR2 protein stability by the HECT E3 ubiquitin ligase Smurf2, and its role in breast cancer progression

Abstract Background Smurf2 E3 ubiquitin ligase physically associates with and regulate the stability of distinct cellular protein substrates. The multi-functional scaffold protein Connector enhancer of kinase suppressor of ras 2 (CNKSR2) plays a key role in regulating cell proliferation, and differentiation through multiple receptor tyrosine kinase pathways. The aim of this study was to investigate whether the interaction between Smurf2 and CNKSR2 has any significant role in the post transcriptional regulation of CNKSR2 expression in breast cancer. Methods: Here we demonstrate a novel interaction of CNKSR2 with Smurf2 by co-immunoprecipitation, indirect immunofluorescence studies, and surface plasmon resonance (SPR) analysis, which can ubiquitinate, but stabilize CNKSR2 by protecting it from proteasome mediated degradation. Results CNKSR2 protein levels were significantly increased upon forced overexpression of Smurf2, indicating the role of Smurf2 in regulating the stability of CNKSR2. Conversely, Smurf2 knockdown resulted in a marked decrease in the protein level expression of CNKSR2 by facilitating enhanced polyubiquitination and proteasomal degradation and reduced the proliferation and clonogenic survival of MDA-MB-231 breast cancer cell lines. Tissue microarray data from 84 patients with various stages of mammary carcinoma, including (in order of increasing malignant potential) normal, usual hyperplasia, fibrocystic changes, fibroadenoma, carcinoma-in-situ, and invasive ductal carcinoma showed a statistically significant association between Smurf2 and CNKSR2 expression, which is also well correlated with the ER, PR, and HER2 status of the tissue samples. A comparatively high expression of Smurf2 and CNKSR2 was observed when the expression of ER and PR was low, and HER2 was high. Consistently, both Smurf2 and CNKSR2 showed an integrated expression in MCF10 breast progression model cell lines. Conclusions Altogether, our findings reveal that Smurf2 is a novel positive regulator of CNKSR2 and suggest that Smurf2-CNKSR2 interaction may serve as a common strategy to control proliferation of human breast cancer cells by modulating CNKSR2 protein stability

Clinical features in patients with CVD.

<p>*Percentages were taken from the column totals.</p

Association between <i>FoxC2</i> genotypes (novel) and CVD risk.

<p>*Percentages were taken from the column totals. Chi-square test for measure of association was used to derive p value. ^aOdds ratio and 95% confidence intervals of individual polymorphisms. ^bAdjusted odds ratio and 95% confidence intervals is obtained adjusting for age group and sex in multiple logistic regression model.</p

Challenges affecting the implementation of e-procurement in the Malawi Public Sector: - The Case of Malawi Housing Corporation, Lilongwe City Council, Kamuzu Central Hospital, Immigration Department and Malawi Defence Force.

As part of e-government initiative, the public sector is encouraged to adopt e-procurement systems and some initiatives are currently underway in Malawi public sector. Despite the numerous benefits of e-procurement and goals set by the Government of Malawi (GoM) to reduce costs, improve service delivery, enhance transparency and efficiency, the public sector is reluctant to move procurement to the Internet and government ministries or departments have not yet implemented e-procurement. This study aimed at assessing the challenges that affect the implementation of e-procurement, perceived benefits, and critical success factors for effective implementation. Both Qualitative and Quantitative research methodologies were used as a guide to achieve the research objectives and the Questionnaire was used to collect data from government ministries and departments. Systematic Random sampling was used to select the government offices to be included in the study then purposive sampling was used to obtain the required primary data from respondents that are knowledgeable with the subject area. Out of the 113 questionnaires distributed, only 91 were received and analyzed, representing 81 percent response rate. Data was analyzed using thematic areas for qualitative (descriptive data) and excel for quantitative data. The results indicate that all the public entities in this study currently do not use any form of e-procurement but they are ready to adopt some. However, the respondents were able to identify the benefits that include, reduced maverick buying and reduced lead time. Challenges for implementation include lack of trained personnel, organizational culture that does not embrace technology, lack of managerial support and cost for setting up the system. The respondents further identified some critical success factors for e-procurement implementation and these include enhanced managerial support and end user training as the major ones. While system integration, users and supplier involvement are the secondary factors for consideration too. The researcher concluded that e-procurement requires resources, training and enactment of law within the public procurement framework for improved efficiency and service delivery. Hence, recommendations have been made to various stakeholders to enhance the implementation process. For instance, the Government of Malawi (GoM) to reduce taxes on ICT equipment and develop curriculum on e-procurement in institutions of higher learning. Executive management of public institutions to involve staff during implementation and train staff on e-procurement systems and tools usage. Lastly, recommendations to employees to acquire the necessary skills through attendance of short term or long term learning from institutions such as Malawi institute of Management (MIM) to enhance computer skills capacity especially in e-procurement

<i>FoxC2</i> based regulation of <i>Hey2</i>, <i>Dll4</i>, <i>COUP TFII</i> and <i>Ephrin B4</i> expression in EA.hy926 cells.

<p><b>(A)</b> Gel image showing increased expression of <i>Dll4</i> and <i>Hey2</i> mRNA in EA.hy926 cells transfected with <i>FoxC2</i> –pCAGIG. Lane 1: 100 bp molecular ladder, Lane 2, 4, 6, 8: <i>FoxC2, GAPDH, Dll4</i> and <i>Hey2</i> mRNA levels in cells transfected with pGAGIG control vector. Lane 3, 5, 7, 9: <i>FoxC2, GAPDH, Dll4</i> and <i>Hey2</i> mRNA levels in <i>FoxC2</i>- pGAGIG construct transfected cells. <b>(B)</b> Gel image showing increased expression of <i>COUP TFII</i> and <i>Ephrin B4</i> mRNA in EA.hy926 cells transfected with <i>FoxC2</i> –pCAGIG. Lane 1, 3, 5: <i>Ephrin B4, COUP TFII</i> and <i>GAPDH</i> mRNA levels in cells transfected with empty pGAGIG control vector. Lane 2, 4, 6: <i>Ephrin B4, COUP TFII</i> and <i>GAPDH</i> mRNA levels in <i>FoxC2</i>- pGAGIG construct transfected cells, Lane 7: 100 bp molecular ladder <b>(C)</b> Relative mRNA levels of <i>Hey2</i> and <i>Dll4</i> measured by qRT-PCR. <b>(D)</b> Relative mRNA levels of <i>COUP TFII</i> and <i>Ephrin B4</i> measured by qRT-PCR. Values are mean±s.d. of triplicate experiments from 3 samples per each group. Statistical significance was determined by Student's t-test (*p<0.05 versus control).</p

Association of allelic frequencies of <i>FoxC2</i> polymorphisms in patients with CVD and control subjects.

<p>*Percentages were taken from the column totals. Chi-squared test for measure of association was used to derive p value.</p

<i>Forkhead box C2</i> Promoter Variant c.-512C>T Is Associated with Increased Susceptibility to Chronic Venous Diseases

<div><p>Chronic venous disease (CVD) is one of the most prevalent yet underrated disorders worldwide. High heritability estimates of CVD indicate prominent genetic components in its etiology and pathology. Mutations in human forkhead box C2 (<i>FoxC2</i>) gene are strongly associated with valve failure in saphenous and deep veins of lower extremities. We explored the association of genetic variants of <i>FoxC2</i> as well as <i>FoxC2</i> mRNA and protein expression levels with CVD of lower limbs. We systematically sequenced the single coding exon, 5′ and 3′ flanking regions of <i>FoxC2</i> gene in 754 study subjects which includes 382 patients with CVD and 372 healthy subjects. Four novel and three reported polymorphisms were identified in our cohort. Three variants in 5′ flanking region and one in 3′ flanking region of <i>FoxC2</i> gene were significantly associated with CVD risk. <i>FoxC2</i> mRNA in vein tissues from 22 patients was 4±1.42 fold increased compared to saphenous veins from 20 normal subjects (p<0.01). FoxC2 protein was also significantly upregulated in varicose veins compared to control samples. The c.-512C>T (<i>rs34221221: C>T</i>) variant which is located in the <i>FoxC2</i> putative promoter region was further analyzed. Functional analysis of c.-512C>T revealed increased mRNA and protein expression in patients with homozygous TT genotype compared to heterozygous CT and wild CC genotypes. Luciferase assay indicated higher transcriptional activity of mutant compared to wild genotype of this variant. These findings suggested that c.-512C>T variant of <i>FoxC2</i> was strongly associated with susceptibility to CVD and also that this variant resulted in FoxC2 overexpression. To obtain a mechanistic insight into the role of upregulated FoxC2 in varicosities, we overexpressed <i>FoxC2</i> in venous endothelial cells and observed elevated expression of arterial markers <i>Dll4</i> and <i>Hey2</i> and downregulation of venous marker <i>COUP-TFII</i>. Our study indicates altered FoxC2-Notch signaling in saphenous vein wall remodeling in patients with varicose veins.</p></div

Effect of <i>FoxC2</i> variant c.-512C>T (rs34221221) on luciferase expression in EA.hy926 cells.

<p>Cell lines were transiently transfected with the FoxC2 promoter reporter plasmid containing CC genotype or TT genotype. The renilla luciferase plasmid was co-transfected with reporter constructs to normalize the luciferase activity. Transfection with mutant construct (pGL4-TT) demonstrated a slight increase in luciferase activity (*p = 0.051) compared to wild pGL4-CC construct. Data shown are the mean ± SD of technical triplicates of two biological experiments.</p

Photomicrograph demonstrating immunohistochemical staining with FoxC2 antibody in venous tissue sections from patients with varicose veins and controls.

<p><b>(A)</b> Negative staining control in normal saphenous veins and <b>(B)</b> varicose vein tissues. <b>(C)</b> FoxC2 expression is localized to tunica media in normal venous tissue section. <b>(D)</b> FoxC2 is expressed all over intima and media of varicose vein tissues with high staining intensity, original magnification x 400.</p