Regulated Intramembrane Proteolysis Research Articles

Trop2: from development to disease.

Trop2 was first discovered as a biomarker of invasive trophoblast cells. Since then most research has focused on its role in tumourigenesis because it is highly expressed in the vast majority of human tumours and animal models of cancer. It is also highly expressed in stem cells and in many organs during development. We review the multifaceted role of Trop2 during development and tumourigenesis, including its role in regulating cell proliferation and migration, self-renewal, and maintenance of basement membrane integrity. We discuss the evolution of Trop2 and its related protein Epcam (Trop1), including their distinct roles. Mutation of Trop2 leads to gelatinous drop-like corneal dystrophy, whereas over-expression of Trop2 in human tumours promotes tumour aggressiveness and increases mortality. Although Trop2 expression is sufficient to promote tumour growth, the surprising discovery that Trop2-null mice have an increased risk of tumour development has highlighted the complexity of Trop2 signaling. Recently, studies have begun to identify the mechanisms underlying TROP2’s functions, including regulated intramembrane proteolysis or specific interactions with integrin b1 and claudin proteins. Understanding the mechanisms underlying TROP2 signaling will clarify its role during development, aid in the development of better cancer treatments and unlock a promising new direction in regenerative medicine.

Cytoplasmic Fragment of Alcadein α Generated by Regulated Intramembrane Proteolysis Enhances Amyloid β-Protein Precursor (APP) Transport into the Late Secretory Pathway and Facilitates APP Cleavage

The neural type I membrane protein Alcadein α (Alcα), is primarily cleaved by amyloid β-protein precursor (APP) α-secretase to generate a membrane-associated carboxyl-terminal fragment (Alcα CTF), which is further cleaved by γ-secretase to secrete p3-Alcα peptides and generate an intracellular cytoplasmic domain fragment (Alcα ICD) in the late secretory pathway. By association with the neural adaptor protein X11L (X11-like), Alcα and APP form a ternary complex that suppresses the cleavage of both Alcα and APP by regulating the transport of these membrane proteins into the late secretory pathway where secretases are active. However, it has not been revealed how Alcα and APP are directed from the ternary complex formed largely in the Golgi into the late secretory pathway to reach a nerve terminus. Using a novel transgenic mouse line expressing excess amounts of human Alcα CTF (hAlcα CTF) in neurons, we found that expression of hAlcα CTF induced excess production of hAlcα ICD, which facilitated APP transport into the nerve terminus and enhanced APP metabolism, including Aβ generation. In vitro cell studies also demonstrated that excess expression of Alcα ICD released both APP and Alcα from the ternary complex. These results indicate that regulated intramembrane proteolysis of Alcα by γ-secretase regulates APP trafficking and the production of Aβ in vivo.

Luman is involved in osteoclastogenesis through the regulation of DC-STAMP expression, stability and localization

ABSTRACTLuman (also known as CREB3) is a type-II transmembrane transcription factor belonging to the OASIS family that localizes to the endoplasmic reticulum (ER) membrane under normal conditions. In response to ER stress, OASIS-family members are subjected to regulated intramembrane proteolysis (RIP), following which the cleaved N-terminal fragments translocate to the nucleus. In this study, we show that treatment of bone marrow macrophages (BMMs) with cytokines – macrophage colony-stimulating factor (M-CSF) and RANKL (also known as TNFSF11) – causes a time-dependent increase in Luman expression, and that Luman undergoes RIP and becomes activated during osteoclast differentiation. Small hairpin (sh)RNA-mediated knockdown of Luman in BMMs prevented the formation of multinucleated osteoclasts, concomitant with the suppression of DC-STAMP, a protein that is essential for cell–cell fusion in osteoclastogenesis. The N-terminus of Luman facilitates promoter activity of DC-STAMP, resulting in upregulation of DC-STAMP expression. Furthermore, Luman interacts with DC-STAMP, and controls its stability and localization. These results suggest that Luman regulates the multinucleation of osteoclasts by promoting cell fusion of mononuclear osteoclasts through DC-STAMP induction and intracellular distribution during osteoclastogenesis.

Regulated Intramembrane Proteolysis of the Colony-Stimulating Factor 1 Receptor during Macrophage Activation

Regulated Intramembrane Proteolysis of the Colony-Stimulating Factor 1 Receptor during Macrophage Activation

EpCAM and the biology of hepatic stem/progenitor cells.

Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein, which is frequently and highly expressed on carcinomas, tumor-initiating cells, selected tissue progenitors, and embryonic and adult stem cells. During liver development, EpCAM demonstrates a dynamic expression, since it can be detected in fetal liver, including cells of the parenchyma, whereas mature hepatocytes are devoid of EpCAM. Liver regeneration is associated with a population of EpCAM-positive cells within ductular reactions, which gradually lose the expression of EpCAM along with maturation into hepatocytes. EpCAM can be switched on and off through a wide panel of strategies to fine-tune EpCAM-dependent functional and differentiative traits. EpCAM-associated functions relate to cell–cell adhesion, proliferation, maintenance of a pluripotent state, regulation of differentiation, migration, and invasion. These functions can be conferred by the full-length protein and/or EpCAM-derived fragments, which are generated upon regulated intramembrane proteolysis. Control by EpCAM therefore not only depends on the presence of full-length EpCAM at cellular membranes but also on varying rates of the formation of EpCAM-derived fragments that have their own regulatory properties and on changes in the association of EpCAM with interaction partners. Thus spatiotemporal localization of EpCAM in immature liver progenitors, transit-amplifying cells, and mature liver cells will decisively impact the regulation of EpCAM functions and might be one of the triggers that contributes to the adaptive processes in stem/progenitor cell lineages. This review will summarize EpCAM-related molecular events and how they relate to hepatobiliary differentiation and regeneration.

Prostaglandin D2 synthase/GPR44: a signaling axis in PNS myelination.

Neuregulin 1 type III is processed following regulated intramembrane proteolysis, which allows communication from the plasma membrane to the nucleus. We found that the intracellular domain of neuregulin 1 type III upregulated the prostaglandin D2 synthase (L-pgds, also known as Ptgds) gene, which, together with the G protein-coupled receptor Gpr44, forms a previously unknown pathway in PNS myelination. Neuronal L-PGDS is secreted and produces the PGD2 prostanoid, a ligand of Gpr44. We found that mice lacking L-PGDS were hypomyelinated. Consistent with this, specific inhibition of L-PGDS activity impaired in vitro myelination and caused myelin damage. Furthermore, in vivo ablation and in vitro knockdown of glial Gpr44 impaired myelination. Finally, we identified Nfatc4, a key transcription factor for myelination, as one of the downstream effectors of PGD2 activity in Schwann cells. Thus, L-PGDS and Gpr44 are previously unknown components of an axo-glial interaction that controls PNS myelination and possibly myelin maintenance.

Sustained induction of collagen synthesis by TGF-β requires regulated intramembrane proteolysis of CREB3L1.

CREB3L1 (cAMP response element binding protein 3-like 1), a transcription factor synthesized as a membrane-bound precursor and activated through Regulated Intramembrane Proteolysis (RIP), is essential for collagen production by osteoblasts during bone development. Here, we show that TGF-β (transforming growth factor-β), a cytokine known to stimulate production of collagen during wound healing and fibrotic diseases, induces proteolytic activation of CREB3L1 in human A549 cells. This activation results from inhibition of expression of TM4SF20 (transmembrane 4 L6 family member 20), which normally inhibits RIP of CREB3L1. Cleavage of CREB3L1 releases its NH2-terminal domain from membranes, allowing it to enter the nucleus where it binds to Smad4 to activate transcription of genes encoding proteins required for assembly of collagen-containing extracellular matrix. Our findings raise the possibility that inhibition of RIP of CREB3L1 could prevent excess deposition of collagen in certain fibrotic diseases.

Evidence of a bacterial receptor for lysozyme: binding of lysozyme to the anti-σ factor RsiV controls activation of the ecf σ factor σV.

σ factors endow RNA polymerase with promoter specificity in bacteria. Extra-Cytoplasmic Function (ECF) σ factors represent the largest and most diverse family of σ factors. Most ECF σ factors must be activated in response to an external signal. One mechanism of activation is the stepwise proteolytic destruction of an anti-σ factor via Regulated Intramembrane Proteolysis (RIP). In most cases, the site-1 protease required to initiate the RIP process directly senses the signal. Here we report a new mechanism in which the anti-σ factor rather than the site-1 protease is the sensor. We provide evidence suggesting that the anti-σ factor RsiV is the bacterial receptor for the innate immune defense enzyme, lysozyme. The site-1 cleavage site is similar to the recognition site of signal peptidase and cleavage at this site is required for σV activation in Bacillus subtilis. We reconstitute site-1 cleavage in vitro and demonstrate that it requires both signal peptidase and lysozyme. We demonstrate that the anti-σ factor RsiV directly binds to lysozyme and muramidase activity is not required for σV activation. We propose a model in which the binding of lysozyme to RsiV activates RsiV for signal peptidase cleavage at site-1, initiating proteolytic destruction of RsiV and activation of σV. This suggests a novel mechanism in which conformational change in a substrate controls the cleavage susceptibility for signal peptidase. Thus, unlike other ECF σ factors which require regulated intramembrane proteolysis for activation, the sensor for σV activation is not the site-1 protease but the anti-σ factor.

Neurotrophins regulate ApoER2 proteolysis through activation of the Trk signaling pathway

BackgroundApoER2 and the neurotrophin receptors Trk and p75NTR are expressed in the CNS and regulate key functional aspects of neurons, including development, survival, and neuronal function. It is known that both ApoER2 and p75NTR are processed by metalloproteinases, followed by regulated intramembrane proteolysis. TrkA activation by nerve growth factor (NGF) increases the proteolytic processing of p75NTR mediated by ADAM17. Reelin induces the sheeding of ApoER2 ectodomain depending on metalloproteinase activity. However, it is not known if there is a common regulation mechanism for processing these receptors.ResultsWe found that TrkA activation by NGF in PC12 cells induced ApoER2 processing, which was dependent on TrkA activation and metalloproteinases. NGF-induced ApoER2 proteolysis was independent of mitogen activated protein kinase activity and of phosphatidylinositol-3 kinase activity. In contrast, the basal proteolysis of ApoER2 increased when both kinases were pharmacologically inhibited. The ApoER2 ligand reelin regulated the proteolytic processing of its own receptor but not of p75NTR. Finally, in primary cortical neurons, which express both ApoER2 and TrkB, we found that the proteolysis of ApoER2 was also regulated by brain-derived growth factor (BDNF).ConclusionsOur results highlight a novel relationship between neurotrophins and the reelin-ApoER2 system, suggesting that these two pathways might be linked to regulate brain development, neuronal survival, and some pathological conditions.

218P - Truncated Epcam is Associated with a More Aggressive Phenotype and Predicts Poor Overall Survival in Colorectal Cancer

ABSTRACT Aim: Regulated intramembrane proteolysis (RIP) has been shown to be an important mechanism for oncogenic activation of EpCAM through nuclear translocation of the intracellular domain EpICD and consequently upregulation of c-myc. Recently, we identified two different membranous EpCAM variants namely EpCAMMF and EpCAMMT to be expressed in the majority of human epithelial carcinomas. The aim of our study was to evaluate the potential of these two candidate protein biomarkers for identification of aggressive colorectal cancer. Methods: Using immunohistochemistry, we analyzed the expression of EpCAMMF and EpCAMMT variants in 632 patients with colorectal cancer and determined their correlations with other prognostic factors and clinical outcome. Results: A statistically significant association was observed for EpCAMMT with advanced tumor stage (p Conclusions: In conclusion, this study indicates for the first time that expression of EpCAMMT is associated with a more aggressive phenotype and predicts poor survival in patients with colorectal cancer. Disclosure: All authors have declared no conflicts of interest.

Regulated Intramembrane Proteolysis of the TGFβ Type I Receptor Conveys Oncogenic Signals

Cancer cells produce high levels of TGFβ, a multipotent cytokine. Binding of TGFβ to its cell surface receptors, the transmembrane serine/threonine kinases TβRII and TβRI, causes phosphorylation and activation of intracellular latent Smad transcription factors. Nuclear Smads act in concert with specific transcription factors to reprogram epithelial cells to become invasive mesenchymal cells. TGFβ also propagates non-canonical signals, so it is crucial to have a better understanding of the underlying molecular mechanisms which favor this pathway. Here we highlight our recent discovery that TGFβ promotes the proteolytic cleavage of TβRI in cancer cells, resulting in the liberation and nuclear translocation of its intracellular domain, acting as co-regulator to transcribe pro-invasive genes. This newly identified oncogenic TGFβ pathway resembles the Notch signaling pathway. We discuss our findings in relation to Notch and provide a short overview of other growth factors that transduce signals via nuclear translocation of their cell surface receptors.

Substrate discrimination by size-exclusion in the intramembrane protease RseP

Regulated intramembrane proteolysis (RIP), wherein a target membrane protein is specifically cleaved within the transmembrane region, is now accepted as a form of cellular signaling. As a result of proteolysis, a soluble portion of the target membrane protein is liberated to act as a signaling molecule. RIP is catalyzed by intramembrane-cleaving proteases, which are now classified into site-2 protease, rhomboid and γ-secretase/SPP families based on the mechanism of catalysis. E. coli possesses a site-2 protease homolog RseP, which is implicated in the extracytoplasmic stress response. RseP cleaves a membrane-spanning anti-σE protein RseA to release σE from the membrane, where truncation of the C-terminal periplasmic part of RseA by a membrane-anchored protease DegS triggers the action of RseP. Hence, there must be some mechanism by which RseP senses the DegS-cleavage of RseA. RseP possesses two tandemly-arranged PDZ domains (PDZ tandem) in the periplasmic region, which have been suggested to be involved in the regulation of cleavage. Although PDZ domains generally recognize the C-terminal sequence of a ligand, most of the previous works suggested that the RseP PDZ domains are involved in the suppression of the intramembrane cleavage of RseA. In this study, we determined the 3D structure of the PDZ tandem by X-ray crystallography and SAXS and showed that the two PDZ domains are arranged in an overall "clam-like" configuration to constitute a "pocket-like" structure. Sequence analysis suggested that the PDZ tandem would lie just above the active center sequestrated within the membrane. Furthermore, chemical modification demonstrated that the interior of the pocket is inaccessible to a bulky reagent in the full-length RseP. Taken together, we have made a proposal that RseP accommodates the truncated RseA into the active center by a steric size-exclusion mechanism through the PDZ tandem, rather than by recognition of a specific sequence/motif of RseA.

Regulated proteolysis of NOTCH2 and NOTCH3 receptors by ADAM10 and presenilins.

In mammals, there are four NOTCH receptors and five Delta-Jagged-type ligands regulating many aspects of embryonic development and adult tissue homeostasis. NOTCH proteins are type I transmembrane receptors that interact with ligands on adjacent cells and are activated by regulated intramembrane proteolysis (RIP). The activation mechanism of NOTCH1 receptors upon ligand binding is well understood and requires cleavage by ADAM10 metalloproteases prior to intramembranous cleavage by γ-secretase. How the other human NOTCH receptor homologues are activated upon ligand binding is not known. Here, we dissect the proteolytic activation mechanism of the NOTCH2 and NOTCH3 receptors. We show that NOTCH2 and NOTCH3 signaling can be triggered by both Delta-Jagged-type ligands and requires ADAM10 and presenilin-1 or -2. Importantly, we did not find any role for the highly related ADAM17/TACE (tumor necrosis factor alpha-converting enzyme) protease in ligand-induced NOTCH2 or NOTCH3 signaling. These results demonstrate that canonical ligand-induced proteolysis of the NOTCH1, -2, and -3 receptors strictly depends on consecutive cleavage of these receptors by ADAM10 and the presenilin-containing γ-secretase complex, leading to transcriptional activation.

Regulated Intramembrane Proteolysis of the Frontotemporal Lobar Degeneration Risk Factor, TMEM106B, by Signal Peptide Peptidase-like 2a (SPPL2a)

The sequential processing of single pass transmembrane proteins via ectodomain shedding followed by intramembrane proteolysis is involved in a wide variety of signaling processes, as well as maintenance of membrane protein homeostasis. Here we report that the recently identified frontotemporal lobar degeneration risk factor TMEM106B undergoes regulated intramembrane proteolysis. We demonstrate that TMEM106B is readily processed to an N-terminal fragment containing the transmembrane and intracellular domains, and this processing is dependent on the activities of lysosomal proteases. The N-terminal fragment is further processed into a small, rapidly degraded intracellular domain. The GxGD aspartyl proteases SPPL2a and, to a lesser extent, SPPL2b are responsible for this intramembrane cleavage event. Additionally, the TMEM106B paralog TMEM106A is also lysosomally localized; however, it is not a specific substrate of SPPL2a or SPPL2b. Our data add to the growing list of proteins that undergo intramembrane proteolysis and may shed light on the regulation of the frontotemporal lobar degeneration risk factor TMEM106B.

Structural basis of DDB1-and-Cullin 4-associated Factor 1 (DCAF1) recognition by merlin/NF2 and its implication in tumorigenesis by CD44-mediated inhibition of merlin suppression of DCAF1 function.

Merlin, a tumor suppressor encoded by the neurofibromatosis type 2 gene, has been shown to suppress tumorigenesis by inhibiting the Cullin 4-RING E3 ubiquitin ligase CRL4(DCAF) (1) in the nucleus. This inhibition is mediated by direct binding of merlin to DDB1-and-Cullin 4-associated Factor 1 (DCAF1), yet the binding mode of merlin to DCAF1 is not well defined. Here, we report structural and biophysical studies of the merlin binding to DCAF1 and its interference with CD44 binding. The crystal structure of the merlin FERM domain bound to the DCAF1 C-terminal acidic tail reveals that the hydrophobic IILXLN motif located at the C-terminal end of DCAF1 binds subdomain C of the FERM domain by forming a β-strand. The binding site and mode resemble that of merlin binding to the CD44 cytoplasmic tail. Competition binding assay showed that CD44 and DCAF1 compete for binding to the merlin FERM domain in solution. The CD44 cytoplasmic tail is known to be cleaved for nuclear translocation by regulated intra-membrane proteolysis (RIP). Our structure implies that, in the nucleus, the CD44 cytoplasmic tail cleaved by RIP could release DCAF1 from merlin by competing for binding to the merlin FERM domain, which results in the inhibition of merlin-mediated suppression of tumorigenesis.

A Histidine-rich Linker Region in Peptidylglycine α-Amidating Monooxygenase Has the Properties of a pH Sensor

Decreasing luminal pH is thought to play a role in the entry of newly synthesized and endocytosed membrane proteins into secretory granules. The two catalytic domains of peptidylglycine α-amidating monooxygenase (PAM), a type I integral membrane protein, catalyze the sequential reactions that convert peptidyl-Gly substrates into amidated products. We explored the hypothesis that a conserved His-rich cluster (His-Gly-His-His) in the linker region connecting its two catalytic domains senses pH and affects PAM trafficking by mutating these His residues to Ala (Ala-Gly-Ala-Ala; H3A). Purified recombinant wild-type and H3A linker peptides were examined using circular dichroism and tryptophan fluorescence; mutation of the His cluster largely eliminated its pH sensitivity. An enzymatically active PAM protein with the same mutations (PAM-1/H3A) was expressed in HEK293 cells and AtT-20 corticotrope tumor cells. Metabolic labeling followed by immunoprecipitation revealed more rapid loss of newly synthesized PAM-1/H3A than PAM-1; although release of newly synthesized monofunctional PHM/H3A was increased, release of soluble bifunctional PAM/H3A, a product of the endocytic pathway, was decreased. Surface biotinylation revealed rapid loss of PAM-1/H3A, with no detectable return of the mutant protein to secretory granules. Consistent with its altered endocytic trafficking, little PAM-1/H3A was subjected to regulated intramembrane proteolysis followed by release of a small nuclear-targeted cytosolic fragment. AtT-20 cells expressing PAM-1/H3A adopted the morphology of wild-type AtT-20 cells; secretory products no longer accumulated in the trans-Golgi network and secretory granule exocytosis was more responsive to secretagogue.

Visualization of Specific γ-Secretase Complexes using Bimolecular Fluorescence Complementation

γ-Secretase is involved in the regulated intramembrane proteolysis of amyloid-β protein precursor (AβPP) and of many other important physiological substrates. γ-secretase is a multiproteic complex made of four main core components, namely presenilin 1 or 2, APH-1, PEN-2, and Nicastrin. Since APH-1 exists as different variants, combinations of these proteins can theoretically yield distinct γ-secretase complexes. Whether γ-secretase complexes trafficking and targeting to either similar or distinct subcellular compartments depend upon their molecular composition remains unknown. A differential complex-specific distribution may drive a narrow specificity for a subset of substrates that would traffic within the same cellular compartments. Here, we generated bigenic expression vectors to co-express untagged nicastrin or presenilin 1 together with either PEN-2 or distinct variants of APH-1 (aL, aS and b) tagged with complementary fragments of the fluorescent protein Venus. We show that these constructs allow the formation of functional γ-secretase complexes and their visualization with bimolecular fluorescence complementation (BiFC). BiFC can be detected at the plasma membrane as well as in endosomes/lysosomes in addition to the endoplasmic reticulum (ER) of COS-7 cells transfected with the different variants of APH-1. However, the majority of cells co-transfected with APH-1b presented BiFC signal only in the ER, suggesting enhanced retention/retrieval of APH-1b-containing γ-secretase complexes. Therefore, the new tools described here should be helpful to decipher the precise subcellular trafficking of γ-secretase complexes and to delineate the distinct variant-linked pathways in various cellular systems.

Single-Molecule Super-Resolution Imaging of TcpP Dynamics in Vibrio Cholerae in Response to Virulence Pathway Deactivation by Increased Cell Density

The agent of the epidemic human disease cholera is Vibrio cholerae, a bacterium which produces cholera toxin via a virulence pathway that has previously been described biochemically. Various environmental stimuli affect this pathway, which is halted when the cell density is low. One virulence pathway component, TcpP, is a transcription activator that acts in concert with other membrane proteins, ToxS, TcpH and ToxR to regulate ToxT expression. TcpP regulation is itself mediated by various cellular signaling pathways. One popular model for the life of each TcpP molecule suggests that TcpP is coupled to a complex made up of ToxR, TcpH and DNA during transcription, and that TcpP is decoupled from this group upon the deactivation of the virulence pathway before degradation by regulated intramembrane proteolysis. This volume change of the diffusing TcpP particle should cause a measurable change in the rate of diffusion as predicted by the Einstein-Stokes equation. Here, we use single-molecule super-resolution fluorescence microscopy to measure the motion of individual TcpP molecules labeled with the photo-activatable fluorescent protein PAmCherry in live V. cholerae cells. Furthermore, we prepare our samples for imaging within an agarose microfluidic device, in which we set up a linear gradient of spent media inside to simulate increasing cell density. By imaging single cells at different signal concentrations on a single slide, variations in preparation or initial cell state will be decoupled from the effect of cell density on the mobility of TcpP, and we observe changes in TcpP-PAmCherry diffusion that we explain in the context of differences in the virulence pathway activity level.

Abstract P2-10-07: Ep-ICD overexpression associates with poor prognosis in invasive ductal carcinoma

Abstract Background: Despite improvements in treatment strategies recurrence rates are still high among breast cancer patients. This may be attributed to heterogeneous nature of breast cancers representing varied morphologic and biological features, behavior, and response to therapy. Even among breast tumors of similar histologic type and grade, prognosis varies. Currently, breast cancer prognosis assessment methods have limited accuracy, are expensive, and in 20-30% of cases lead to over-treatment with adverse effects. None of the currently known prognostic factors has the ability to predict accurately which breast cancer patients are at high risk of recurrence. Thus, there is an increasing need for identification and validation of prognostic markers for assessment of risk for disease recurrence in breast cancer patients. Epithelial cell adhesion molecule (EpCAM) is a glycosylated, 30- to 40-kDa type I membrane protein, expressed in several human epithelial tissues and overexpressed in cancers, as well as in progenitors, normal and cancer stem cells, and is implicated in epithelial mesenchymal transition (EMT). Regulated intra-membrane proteolysis (RIP) of EpCAM by tumor-necrosis-factor alpha converting enzyme (TACE) results in shedding of its extracellular domain (EpEx) and release of intracellular domain, Ep-ICD, into the cytoplasm. Ep-ICD can signal into the cell nucleus by engagement of components of the Wnt pathway proteins including four and one half LIM domains protein 2 (FHL2), β-catenin and Lef, leading to activation of its oncogenic activity. Objective. Evaluate the prognostic significance of Ep-ICD overexpression in invasive ductal carcinoma (IDC). Methodology: Formalin fixed paraffin embedded (FFPE) tissue sections obtained from IDCs (n = 180) and normal breast tissues (n = 45) were used for immunostaining for Ep-ICD using specific monoclonal antibody. A semi-quantitative visual scoring of the immunostaining results for Ep-ICD based on percentage of tumor cells stained and intensity of scoring was used to compare the expression in breast cancers and normal tissues. Statistical analysis was carried out to determine the association of Ep-ICD expression with clinical outcome. Results: Among the 180 IDCs analyzed, nuclear Ep-ICD was observed in 75 tissues (41.7%) while cytoplasmic positivity was observed in 145 tissues (80.6%). In comparison, nuclear Ep-ICD localization was observed only in 11 normal tissues (23.9%) and cytoplasmic positivity was observed in 39 normal tissues (86.7%). The nuclear / cytoplasmic Ep-ICD expression has been correlated with at least 5 years follow-up data of 180 breast cancer patients after primary treatment. Kaplan Meier survival analysis showed significantly reduced 5 year disease free survival in IDC patients showing nuclear positivity (p < 0.001) or cytoplasmic positivity (p = 0.048). In Cox multivariate regression analysis, nuclear Ep-ICD overexpression emerged as an independent indicator of poor prognosis in IDCs (p = 0.008, H.R. = 81.18). Conclusion: Among invasive ductal carcinomas of the breast, nuclear Ep-ICD overexpression predicts reduced 5-year disease free survival. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-10-07.

HER4 selectively coregulates estrogen stimulated genes associated with breast tumor cell proliferation

The EGFR-family member HER4 undergoes regulated intramembrane proteolysis (RIP) to generate an intracellular domain (4ICD) that functions as a transcriptional coactivator. Accordingly, 4ICD coactivates the estrogen receptor (ER) and associates with ER at target gene promoters in breast tumor cells. However, the extent of 4ICD coactivation of ER and the functional significance of the 4ICD/ER transcriptional complex is unclear. To identify 4ICD coactivated genes we performed a microarray gene expression analysis of β-estradiol treated cells comparing control MCF-7 breast cancer cells to MCF-7 cells where HER4 expression was stably suppressed using a shRNA. In the MCF-7 cell line, β-estradiol significantly stimulated or repressed by 2-fold or more 726 or 53 genes, respectively. Significantly, HER4/4ICD was an obligate coactivator for 277 or 38% of the β-estradiol stimulated genes. Ingenuity Pathway Analysis of β-estradiol regulated genes identified significant associations with multiple cellular functions regulating cellular growth and proliferation, cell cycle progression, cancer metastasis, decreased hypoplasia, tumor cell migration, apoptotic resistance of tumor cells, and increased transcription. Genes coactivated by 4ICD displayed functional specificity by only significantly contributing to cellular growth and proliferation, cell cycle progression, and decreased hypoplasia. In direct concordance with these in situ results we show that HER4 knockdown in MCF-7 cells results in a loss of estrogen stimulated tumor cell proliferation and cell cycle progression, whereas, estrogen stimulated tumor cell migration was unaffected by loss of HER4 expression. In summary, we demonstrate for the first time that a cell surface receptor functions as an obligate ER coactivator with functional specificity associated with breast tumor cell proliferation and cell cycle progression. Nearly 90% of ER positive tumors coexpress HER4, therefore we predict that the majority of breast cancer patients would benefit from a strategy to therapeutic disengage ER/4ICD coregulated tumor cell proliferation.