Characterization of Effects of Muc1 Expression on Epidermal Growth Factor Receptor Signaling in Breast Cancer

EGF receptors are key regulators of cell survival and growth in normal and transformed tissues. Ligand binding results in formation of homo/hetero dimers of these receptors, followed by activation of the kinase activity and subsequent tyrosine phosphorylation of many downstream molecules. The activation of these receptors is not only mediated by the binding of their cognate ligands, but by transactivaton by other molecules as well. Recent studies have identified an oncogenic glycoprotein MUC1 as a binding partner for EGFR and that MUC1 expression can potentiate EGFR-dependent signal transduction. After receptor activation, EGFR is typically downregulated via an endocytic pathway that results in receptor degradation or recycling. We report here that MUC1 expression inhibits the degradation of ligand-activated erbB1. In addition, MUC1 expression results in prolonged activation of Akt, but not ERK1,2 MAPKinase. The MUC1-mediated protection against degradation occurs with a decrease in EGF-stimulated ubiquitination of erbB1, and an increase in erbB1 recycling. We then utilized the WAP-TGFα transgenic mouse model of breast cancer and determined that a loss of Muc1 expression dramatically alters mammary tumor progression. While 100% of WAP-TGFα/Muc1^(+/+) mice form mammary gland tumors, only 37% of WAP-TGFα/Muc1^(-/-) form tumors. Furthermore, expression of cyclin D1 expression is significantly suppressed in tumors derived from WAPTGFα/Muc1^(-/-) animals, and loss of Muc1 expression resulted in a significant inhibition in the formation of hyperplastic lesions in the mammary gland. We also observed metastatic pulmonary adenocarcinoma (1/29) and perivascular lymphoma of unknown origin (28/29) in the WAP-TGFα transgenic mice but not in the WAP TGFα/Muc1^(-/-) animals. To determine the effects of Muc1 expression on metastasis in a model lacking perivascular lymphoma, we crossed MMTV-Wnt-1 and MMTV-MUC1 transgenic mice and evaluated interactions between Muc1 and EGFR. Although the MMTV-Wnt-1 mice are non-metastatic, a majority (6/10) of the bitransgenic MMTVWnt- 1/MMTV-MUC1 formed pulmonary metastases. Furthermore, overexpression of MUC1 increases the breast cancer cell invasion in vitro. The MUC1 induced increase in invasion is found to be EGF and EGFR-kinase dependent. Collectively, these data indicate that MUC1 expression contributes to many of the hallmarks of cancer and in addition, is an important modulator of EGFR-associated mammary tumor progression

Kaposi's sarcoma-associated herpesvirus K-Rta exhibits SUMO-targeting ubiquitin ligase (STUbL) like activity and is essential for viral reactivation.

The small ubiquitin-like modifier (SUMO) is a protein that regulates a wide variety of cellular processes by covalent attachment of SUMO moieties to a diverse array of target proteins. Sumoylation also plays an important role in the replication of many viruses. Previously, we showed that Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a SUMO-ligase, K-bZIP, which catalyzes sumoylation of host and viral proteins. We report here that this virus also encodes a gene that functions as a SUMO-targeting ubiquitin-ligase (STUbL) which preferentially targets sumoylated proteins for degradation. K-Rta, the major transcriptional factor which turns on the entire lytic cycle, was recently found to have ubiquitin ligase activity toward a selected set of substrates. We show in this study that K-Rta contains multiple SIMs (SUMO interacting motif) and binds SUMOs with higher affinity toward SUMO-multimers. Like RNF4, the prototypic cellular STUbL, K-Rta degrades SUMO-2/3 and SUMO-2/3 modified proteins, including promyelocytic leukemia (PML) and K-bZIP. PML-NBs (nuclear bodies) or ND-10 are storage warehouses for sumoylated proteins, which negatively regulate herpesvirus infection, as part of the intrinsic immune response. Herpesviruses have evolved different ways to degrade or disperse PML bodies, and KSHV utilizes K-Rta to inhibit PML-NBs formation. This process depends on K-Rta's ability to bind SUMO, as a K-Rta SIM mutant does not effectively degrade PML. Mutations in the K-Rta Ring finger-like domain or SIM significantly inhibited K-Rta transactivation activity in reporter assays and in the course of viral reactivation. Finally, KSHV with a mutation in the Ring finger-like domain or SIM of K-Rta replicates poorly in culture, indicating that reducing SUMO-conjugates in host cells is important for viral replication. To our knowledge, this is the first virus which encodes both a SUMO ligase and a SUMO-targeting ubiquitin ligase that together may generate unique gene regulatory programs

Kaposi's sarcoma-associated herpesvirus K-Rta exhibits SUMO-targeting ubiquitin ligase (STUbL) like activity and is essential for viral reactivation.

The small ubiquitin-like modifier (SUMO) is a protein that regulates a wide variety of cellular processes by covalent attachment of SUMO moieties to a diverse array of target proteins. Sumoylation also plays an important role in the replication of many viruses. Previously, we showed that Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a SUMO-ligase, K-bZIP, which catalyzes sumoylation of host and viral proteins. We report here that this virus also encodes a gene that functions as a SUMO-targeting ubiquitin-ligase (STUbL) which preferentially targets sumoylated proteins for degradation. K-Rta, the major transcriptional factor which turns on the entire lytic cycle, was recently found to have ubiquitin ligase activity toward a selected set of substrates. We show in this study that K-Rta contains multiple SIMs (SUMO interacting motif) and binds SUMOs with higher affinity toward SUMO-multimers. Like RNF4, the prototypic cellular STUbL, K-Rta degrades SUMO-2/3 and SUMO-2/3 modified proteins, including promyelocytic leukemia (PML) and K-bZIP. PML-NBs (nuclear bodies) or ND-10 are storage warehouses for sumoylated proteins, which negatively regulate herpesvirus infection, as part of the intrinsic immune response. Herpesviruses have evolved different ways to degrade or disperse PML bodies, and KSHV utilizes K-Rta to inhibit PML-NBs formation. This process depends on K-Rta's ability to bind SUMO, as a K-Rta SIM mutant does not effectively degrade PML. Mutations in the K-Rta Ring finger-like domain or SIM significantly inhibited K-Rta transactivation activity in reporter assays and in the course of viral reactivation. Finally, KSHV with a mutation in the Ring finger-like domain or SIM of K-Rta replicates poorly in culture, indicating that reducing SUMO-conjugates in host cells is important for viral replication. To our knowledge, this is the first virus which encodes both a SUMO ligase and a SUMO-targeting ubiquitin ligase that together may generate unique gene regulatory programs

STUbL-like function is important for K-Rta transactivation activity.

<p>Reporter assays were performed in 293 cells using the indicated K-Rta target gene reporters. Reporter plasmids were cotransfected with K-Rta Wt or mutants, and luciferase activity was measured at 48 hours post-transfection. Luciferase activity of reporter with empty expression plasmids was normalized to a value of 1. Fold activation over control is shown.</p

Immunofluorescence analysis (IFA).

<p><b> (A)</b> IFA was performed with anti-PML (Green) and anti-K-Rta (Red) antibodies. Arrows indicate cells showing overexpression of K-Rta. <b>(B)</b> K-Rta degrades PML. PML wild type or PML del-SUMO mutant was cotransfected with K-Rta or K-Rta mutants. K-Rta wild type preferentially degrades PML wild type, which can be modified by SUMO in vivo. K-Rta Ring mutant (H145L) or SUMO-binding mutant (<i>Δ</i>SIM) was not able to degrade PML. <b>(C)</b> Endogenous SUMO-modified proteins. K-Rta expression was induced by addition of doxycycline (Dox). Cell lysates were prepared 48 hours post-induction. Indicated proteins were probed with specific antibodies and 25 µg of total protein was loaded in each lane. GAPDH was used as a loading control.</p

K-Rta Wt but not Ring-finger domain mutant can degrade SUMO-modified proteins.

<p>(<b>A</b>) Flag-SUMO and K-Rta wild type or mutant were cotransfected into 293T cells, and probed with indicated antibodies. The K-Rta Ring-like domain (C141, H145) is important for SUMO degradation. (<b>B</b>) MG132 recovered SUMO proteins from degradation. 24 hours after the K-Rta transfection, MG132 or DMSO (vehicle) was added into the culture media and cells were harvested after 12 hours of treatment. SUMO-modified proteins were probed with anti-Flag antibody. (<b>C</b>) SUMO degradation during KSHV reactivation in BCBL-1. After induction of K-Rta expression in TREx-K-Rta-BCBL-1 with doxycycline (Dox), SUMO-modified proteins were probed with anti-Flag antibody. K-Rta induction was confirmed with an anti-K-Rta antibody and GAPDH was served as the loading control. (<b>D</b>) Recovery of SUMO-modified proteins with MG132 in BCBL-1. KSHV reactivation was triggered by induction of K-Rta in BCBL-1 cells in either the presence or absence of MG132. The amount of SUMO-modified proteins was examined by immunoblotting with an anti-Flag antibody. The accumulation of SUMO-modified conjugates was evident by increments of higher molecular weight entities.</p

SUMO-degradation function is important for KSHV replication.

<p><b>(A)</b> Recombinant KSHV, which harbors a point mutation in the Ring domain of K-Rta or SIM domain was constructed and transfected into 293T cells. LANA expression was examined by RT-qPCR (a). Values are normalized to cellular GAPDH. Recombinant KSHV containing a K-Rta point mutation or revertant wild type virus was reactivated with combination of sodium butyrate (SB, 1 mM) and TPA (20 nM). Expression of K-Rta protein was confirmed by immunoblotting (b), and K-Rta target gene expression was examined by RT-qPCR (c). Values are normalized to cellular β-actin, and fold induction over latent cell is shown. <b>(B)</b> Viral replication. Recombinant KSHV replication was measured after reactivation with TPA, sodium butyrate, or combination of K-Rta expression and TPA treatment. KSHV encapsidated (DNase resistant) DNA copy number in the supernatant was measured by qt-PCR. Absolute viral copy number in 1 mL of supernatant is shown. K-Rta Wt but not mutant expression rescued viral replication.</p

K-Rta degrades SUMO(+)K-bZIP but not SUMO(−)K-bZIP.

<p>K-Rta was cotransfected with the indicated K-bZIP plasmid and immunoblotting was performed with anti-K-bZIP antibody. Proteasome (MG132, MG; Epoxomicin, Epox) or lysosome inhibitors (chloroquine, Chlor) were added to the culture media after 24 hours transfection and incubated another 12 hours.</p

Degradation of SUMO by K-Rta.

<p>Plasmids expressing E1 (Uba2/Aos), E2 (Ubc9), SUMO-2, K-Rta or empty vector (Vec) were transfected into 293T cells. SUMO, K-Rta or tubulin was probed with respective antibody. <b>(A)</b> K-Rta reduced total SUMO-modified protein levels in a dose-dependent manner. <b>(B)</b> Overexpression of SUMO but not other SUMO enzymes recover the cellular SUMO-modified proteins.</p