EpCAM was found to be overexpressed on epithelial progenitors, carcinomas and cancer-initiating cells. The role of EpCAM in proliferation, and its association with cancer is poorly explained by proposed cell adhesion functions. Here we show that regulated intramembrane proteolysis activates EpCAM as a mitogenic signal transducer in vitro and in vivo. This involves shedding of its ectodomain EpEX and nuclear translocation of its intracellular domain EpICD. Cleavage of EpCAM is sequentially catalysed by TACE and presenilin-2. Pharmacological inhibition or genetic silencing of either protease impairs growth-promoting signalling by EpCAM, which is compensated for by EpICD. Released EpICD associates with FHL2, beta-catenin and Lef-1 to form a nuclear complex that contacts DNA at Lef-1 consensus sites, induces gene transcription and is oncogenic in immunodeficient mice. In patients, EpICD was found in nuclei of colon carcinoma but not of normal tissue. Nuclear signalling of EpCAM explains how EpCAM functions in cell proliferation.

Epithelial cell adhesion molecule (EpCAM) is a membrane glycoprotein expressed on adenomatous and simple epithelia, where it is involved in homophilic adhesion at the basolateral membrane. Carcinomas strongly overexpress EpCAM through an, as yet, unknown mechanism. Interestingly, otherwise EpCAM-negative squamous epithelia are seen to express EpCAM concomitant with their transformation and de-differentiation. The amount of EpCAM and the number of expressing cells both increase with the grade of dysplasia. Despite an important amount of data correlating the expression of EpCAM with cellular proliferation and de-differentiation, such as the coexpression with Ki-67, a marker for proliferation, it is unknown whether EpCAM may directly contribute to carcinogenesis. Here, we show that EpCAM has a direct impact on cell cycle and proliferation, and the ability to rapidly upregulate the proto-oncogene c-myc and cyclin A/E. Human epithelial 293 cells as well as murine NIH3T3 fibroblasts expressing EpCAM had a decreased requirement for growth factors, enhanced metabolic activity and colony formation capacity. Importantly, the inhibition of EpCAM expression with antisense mRNA led to a strong decrease in proliferation and metabolism in human carcinoma cells. Moreover, domain swapping experiments demonstrated that the intracellular part of EpCAM is necessary and sufficient to transduce the effects described. Thus, the data presented here highlight the role of EpCAM, demonstrating for the first time a direct link to cell cycle and proliferation.

IntroductionB cells play essential roles during protective immune responses to invading pathogens. On encounter of foreign antigen and with cognate T-cell help, B lymphocytes proliferate and form distinct histologic structures, termed germinal center (GC). In the GC, they undergo somatic hypermutation and class-switch recombination. During somatic hypermutation, they introduce random mutations into their immunoglobulin variable regions while they exchange the heavy chain constant region during class-switch recombination to allow for different effector functions. After a selection process by antigen, B cells differentiate into memory B cells and plasma cells (PCs), which secrete antibodies. 1 The deregulation of this process is heavily implicated in human disease. Production of class-switched antibodies against self-antigens causes or contributes to various autoimmune syndromes and unrestrained B-cell proliferation and survival can result in lymphomas. 1,2 It is thought that the majority of human lymphomas derive from the GC, probably because the DNA damage inherent to the GC reaction facilitates mutations and chromosomal translocations. 1,3 Recently, the ubiquitin-editing enzyme A20, encoded by the tumor necrosis factor-␣-inducible gene 3 (TNFAIP3), has been associated with both autoimmunity and lymphomagenesis. Polymorphisms and mutations in or near the TNFAIP3 genomic locus have been linked with various human autoimmune syndromes with a strong humoral component, such as systemic lupus erythematosus (SLE), 4,5 rheumatoid arthritis, 6,7 and celiac disease. 8 Loss of A20 function through mutations, chromosomal deletions, and/or promoter methylation is a frequent event in several human lymphomas, 9-12 all of which are characterized by constitutive activation of nuclear factor-B (NF-B). 13 These factors regulate a plethora of genes encoding for proinflammatory mediators, antiapoptotic proteins, cell adhesion molecules and, for negative feedback control, inhibitory proteins, such as p100, IB␣, and A20. 14,15 During the transmission of NF-B activating signals from cell-surface receptors such as the B-cell receptor (BCR), CD40, or Toll-like receptors (TLRs), signal transduction occurs via the attachment of polyubiquitin chains to key proteins, including MALT1 or TRAF6. Polyubiquitin chains, linked via K63 or linear assembly, serve to recruit different kinase complexes. In the case of canonical NF-B, induced proximity allows the upstream kinase TAK1 to phosphorylate its target IKK2, which then effects NF-B activation. A20, whose transcription is induced by NF-B, dampens signaling through 2 main activities. First, as deubiquitinase A20 removes K63-linked polyubiquitin chains from essential signaling intermediates, such as TRAF6. Second, A20 induces, in concert with other proteins, degradation of some of its molecular targets, through addition of K48-linked ubiquitin chains. 14,16 Degradation of RIP1 limits TNF-induced signaling, 14 whereas degradation of the K63-chain-specific E2 ligases Ubc13/UbcH5c generally affects t...

BackgroundCD44 splice variants are long-known as being associated with cell transformation. Recently, the standard form of CD44 (CD44s) was shown to be part of the signature of cancer stem cells (CSCs) in colon, breast, and in head and neck squamous cell carcinomas (HNSCC). This is somewhat in contradiction to previous reports on the expression of CD44s in HNSCC. The aim of the present study was to clarify the actual pattern of CD44 expression in head and neck epithelia.MethodsExpression of CD44s and CD44v6 was analysed by immunohistochemistry with specific antibodies in primary head and neck tissues. Scoring of all specimens followed a two-parameters system, which implemented percentages of positive cells and staining intensities from − to +++ (score = %×intensity; resulting max. score 300). In addition, cell surface expression of CD44s and CD44v6 was assessed in lymphocytes and HNSCC.ResultsIn normal epithelia CD44s and CD44v6 were expressed in 60–95% and 50–80% of cells and yielded mean scores with a standard error of a mean (SEM) of 249.5±14.5 and 198±11.13, respectively. In oral leukoplakia and in moderately differentiated carcinomas CD44s and CD44v6 levels were slightly increased (278.9±7.16 and 242±11.7; 291.8±5.88 and 287.3±6.88). Carcinomas in situ displayed unchanged levels of both proteins whereas poorly differentiated carcinomas consistently expressed diminished CD44s and CD44v6 levels. Lymphocytes and HNSCC lines strongly expressed CD44s but not CD44v6.ConclusionCD44s and CD44v6 expression does not distinguish normal from benign or malignant epithelia of the head and neck. CD44s and CD44v6 were abundantly present in the great majority of cells in head and neck tissues, including carcinomas. Hence, the value of CD44s as a marker for the definition of a small subset of cells (i.e. less than 10%) representing head and neck cancer stem cells may need revision.

The epithelial cell adhesion molecule (EpCAM) is an integral transmembrane protein that is frequently overexpressed in embryonic stem cells, tissue progenitors, carcinomas and cancer-initiating cells. In cancer cells, expression of EpCAM is associated with enhanced proliferation and upregulation of target genes including c-myc. However, the exact molecular mechanisms underlying the observed EpCAM-dependent cell proliferation remained unexplored. Here, we show that EpCAM directly affects cell cycle progression via its capacity to regulate the expression of cyclin D1 at the transcriptional level and depending on the direct interaction partner FHL2 (four-and-a-half LIM domains protein 2). As a result, downstream events such as phosphorylation of the retinoblastoma protein (Rb) and expression of cyclins E and A are similarly affected. In vivo, EpCAM expression strength and pattern are both positively correlated with the proliferation marker Ki67, high expression and nuclear localisation of cyclin D1, and Rb phosphorylation. Thus, EpCAM enhances cell cycle progression via the classical cyclin-regulated pathway.