Pamela M.J. McLaughlin

EpCAM in carcinogenesis: the good, the bad or the ugly

The epithelial cell adhesion molecule (EpCAM) is a membrane glycoprotein that is highly expressed on most carcinomas and therefore of potential use as a diagnostic and prognostic marker for a variety of carcinomas. Interestingly, EpCAM is explored as target in antibody-based therapies. Recently, EpCAM has been identified as an additional marker of cancer-initiating cells. In this review, we describe the controversial biological role of EpCAM with the focus on carcinogenesis: as an adhesion molecule, EpCAM mediates homophilic adhesion interactions, which in turn might prevent metastasis. On the other hand, EpCAM abrogates E-cadherin mediated cell-cell adhesion thereby promoting metastasis. Also, upon cleavage of EpCAM, the intracellular domain functions as a part of a transcriptional complex inducing c-myc and cyclin A and E. In line with these seemingly controversial roles, EpCAM overexpression has been associated with both decreased and increased survival of patients. Similarly, either induction or downregulation of EpCAM expression lowers the oncogenic potential depending on the cell type. As epigenetic dysregulation underlies aberrant EpCAM expression, we propose epigenetic editing as a novel approach to investigate the biological role of EpCAM, expanding the options for EpCAM as a therapeutic target in cancer.

The ubiquitin‐activating enzyme E1‐like protein in lung cancer cell lines

The UBE1L gene isolated from the chromosome 3p21 region has an extremely reduced level of mRNA in lung cancer. Sequence analysis showed a 45% homology to the human ubiquitin‐activating enzyme E1 at the amino acid level. To further characterize the protein product, we generated UBE1L protein‐specific antibodies. Immunoblot analysis revealed a full‐length gene product of approximately 112 kDa. Assessment of the level and distribution pattern of the UBE1L protein in normal and tumor tissue using the generated antibodies showed that the UBE1L protein was present in normal lung cells and non‐lung cancer cell lines, but was undetectable in all 14 human lung cancer cell lines analyzed. This difference in expression of the UBE1L protein between normal lung tissue and lung tumor‐derived cell lines suggests a possible involvement of an E1‐like protein in the origin and/or progression of lung tumors. Int. J. Cancer 85:871–876, 2000.

CD64-directed immunotoxin inhibits arthritis in a novel CD64 transgenic rat model

Macrophages are known to play a key role during inflammation in rheumatoid arthritis (RA). Inflammatory macrophages have increased expression of CD64, the high-affinity receptor for IgG. Targeting this receptor through a CD64-directed immunotoxin, composed of an Ab against CD64 and Ricin A, results in effective killing of inflammatory macrophages. In this study, we show elevated levels of CD64 on synovial macrophages in both synovial lining and synovial fluid in RA patients. The CD64-directed immunotoxin efficiently eliminates activated synovial macrophages in vitro, while leaving quiescent, low CD64-expressing macrophages unaffected. To examine whether killing of CD64 macrophages results in therapeutic effects in vivo, we established an adjuvant arthritis (AA) model in newly generated human CD64 (hCD64) transgenic rats. We demonstrate that hCD64 regulation in this transgenic rat model is similar as in humans. After AA induction, treatment with CD64-directed immunotoxin results in significant inhibition of disease activity. There is a direct correlation between immunotoxin treatment and decreased macrophage numbers, followed by diminished inflammation and bone erosion in paws of these hCD64 transgenic rats. These data support synovial macrophages to play a crucial role in joint inflammation in AA in rats and in human RA. Selective elimination of inflammatory macrophages through a CD64-directed immunotoxin may provide a novel approach for treatment of RA.

Targeted DNA Methylation by a DNA Methyltransferase Coupled to a Triple Helix Forming Oligonucleotide To Down-Regulate the Epithelial Cell Adhesion Molecule

The epithelial cell adhesion molecule (EpCAM) is a membrane glycoprotein that has been identified as a marker of cancer-initiating cells. EpCAM is highly expressed on most carcinomas, and transient silencing of EpCAM expression leads to reduced oncogenic potential. To silence the EpCAM gene in a persistent manner via targeted DNA methylation, a low activity mutant (C141S) of the CpG-specific DNA methyltransferase M.SssI was coupled to a triple-helix-forming oligonucleotide (TFO−C141S) specifically designed for the EpCAM gene. Reporter plasmids encoding the green fluorescent protein under control of different EpCAM promoter fragments were treated with the TFO−C141S conjugate to determine the specificity of targeted DNA methylation in the context of a functional EpCAM promoter. Treatment of the plasmids with TFO−C141S resulted in efficient and specific methylation of the targeted CpG located directly downstream of the triple helix forming site (TFS). No background DNA methylation was observed neither in a 700 bp region of the EpCAM promoter nor in a 400 bp region of the reporter gene downstream of the TFS. Methylation of the target CpG did not have a detectable effect on promoter activity. This study shows that the combination of a specific TFO and a reduced activity methyltransferase variant can be used to target DNA methylation to predetermined sites with high specificity, allowing determination of crucial CpGs for promoter activity.

Engineering zinc finger protein transcription factors to downregulate the epithelial glycoprotein-2 promoter as a novel anti-cancer treatment

Zinc finger protein transcription factors (ZFP‐TFs) are emerging as powerful novel tools for the treatment of many different diseases. ZFPs are DNA‐binding motifs and consist of modular zinc finger domains. Each domain can be engineered to recognize a specific DNA triplet, and stitching six domains together results in the recognition of a gene‐specific sequence. Inhibition of gene expression can be achieved by fusing a repressor domain to these DNA‐binding motifs. In this study, we engineered ZFP‐TFs to downregulate the activity of the epithelial glycoprotein‐2 (EGP‐2) promoter. The protein EGP‐2 is overexpressed in a wide variety of cancer types and EGP‐2 downregulation has been shown to result in a decreased oncogenic potential of tumor cells. Therefore, downregulation of EGP‐2 expression by ZFP‐TFs provides a novel anti‐cancer therapeutic. Using a straightforward strategy, we engineered a 3‐ZFP that could bind a 9 bp sequence within the EGP‐2 promoter. After the addition of a repressor domain, this 3‐ZFP‐TF could efficiently downregulate EGP‐2 promoter activity by 60%. To demonstrate the flexibility of this technology, we coupled an activation domain to the engineered ZFP, resulting in a nearly 200% increase in EGP‐2 promoter activity. To inhibit the endogenous EGP‐2 promoter, we engineered 6‐ZFP‐TFs. Although none of the constructed ZFP‐TFs could convincingly modulate the endogenous promoter, efficient and specific inhibition of the exogenous promoter was observed. Overall, ZFP‐TFs are versatile bi‐directional modulators of gene expression and downregulation of EGP‐2 promoter activity using ZFP‐TFs can ultimately result in a novel anti‐cancer treatment.

EpCAM in morphogenesis.

Embryonic development is one of the most complex biological phenomena that involves the appropriate expression and synchronized interactions of a plethora of proteins, including cell adhesion molecules (CAMs). Many members of the diverse family of CAMs have been shown to be critically involved in the correct execution of embryonic development. The Epithelial Cell Adhesion Molecule (EpCAM) is an atypical cell adhesion molecule originally identified as a marker for carcinoma. However, recent insights have revealed that EpCAM participates in not only cell adhesion, but also in proliferation, migration and differentiation of cells. All of these processes are known to be fundamental for morphogenesis. Here, we review the current literature that establishes EpCAM as a protein involved in morphogenesis, starting from the earliest stages of embryogenesis and ending in organogenesis. In addition, we provide directions for further elucidation of the role of EpCAM in embryogenesis.

Persistent downregulation of the pancarcinoma-associated epithelial cell adhesion molecule via active intranuclear methylation

The epithelial cell adhesion molecule (EpCAM) is expressed at high levels on the surface of most carcinoma cells. SiRNA silencing of EpCAM expression leads to reduced metastatic potential of tumor cells demonstrating its importance in oncogenesis and tumor progression. However, siRNA therapy requires either sequential delivery or integration into the host cell genome. Hence we set out to explore a more definite form to influence EpCAM gene expression. The mechanisms underlying the transcriptional activation of the EpCAM gene, both in normal epithelial tissue as well as in carcinogenesis, are poorly understood. We show that DNA methylation plays a crucial role in EpCAM expression, and moreover, active silencing of endogenous EpCAM via methylation of the EpCAM promoter results in a persistent downregulation of EpCAM expression. In a panel of carcinoma derived cell lines, bisulfite analyses showed a correlation between the methylation status of the EpCAM promoter and EpCAM expression. Treatment of EpCAM‐negative cell lines with a demethylating agent induced EpCAM expression, both on mRNA and protein level, and caused upregulation of EpCAM expression in an EpCAM‐positive cell line. After delivery of the DNA methyltransferase M.SssI into EpCAM‐positive ovarian carcinoma cells, methylation of the EpCAM promoter resulted in silencing of EpCAM expression. SiRNA‐mediated silencing remained for 4 days, after which EpCAM re‐expression increased in time, while M.SssI‐mediated downregulation of EpCAM maintained through successive cell divisions as the repression persisted for at least 17 days. This is the first study showing that active DNA methylation leads to sustained silencing of endogenous EpCAM expression.

Cancer immunotherapy: insights from transgenic animal models

A wide range of strategies in cancer immunotherapy has been developed in the last decade, some of which are currently being used in clinical settings. The development of these immunotherapeutical strategies has been facilitated by the generation of relevant transgenic animal models. Since the different strategies in experimental immunotherapy of cancer each aim to activate different immune system components, a variety of transgenic animals have been generated either expressing tumor associated, HLA, oncogenic or immune effector cell molecule proteins. This review aims to discuss the existing transgenic mouse models generated to study and develop cancer immunotherapy strategies and the variable results obtained. The potential of the various transgenic animal models regarding the development of anti-cancer immunotherapeutical strategies is evaluated.

Expression of EpCAM is up-regulated during regeneration of renal epithelia†

The human epithelial cell adhesion molecule (hEpCAM) is involved in epithelial morphogenesis and repair of epithelial tissues. We hypothesized that changes in hEpCAM expression in vivo correlate with regeneration of renal epithelia after ischaemia/reperfusion injury (IRi). Unilateral IRi was performed on kidneys of hEpCAM transgenic mice. Changes in hEpCAM expression were investigated by quantitative RT‐PCR in renal cortex and medulla dissected by laser dissection microscopy and expression patterns of hEpCAM in regenerating kidneys were assessed by immunohistochemistry. The mechanism of hEpCAM promoter activation was investigated in vitro, by real‐time bioluminescent imaging in HK‐2 cells and in primary tubular epithelial cells (PTECs) subjected to hypoxia and reoxygenation. In vivo, the transcription of the human epcam gene significantly increased in the renal cortex during tubular re‐epithelialization (p < 0.01). Moreover, the number of tubuli that expressed hEpCAM protein more than doubled in the renal cortex during regeneration. De novo expression of hEpCAM was detected in the S1 segments of proximal tubuli. Under hypoxic conditions in vitro, activity of the hEpCAM promoter was up‐regulated two‐fold in the HK‐2 proximal epithelial cell line. Moreover, both in primary proximal epithelial cells and in HK‐2 cells, hEpCAM protein expression was increased after hypoxia and reoxygenation. The significant up‐regulation of hEpCAM during post‐ischaemic renal regeneration in vivo and during in vitro hypoxia indicates that hEpCAM expression is associated with renal regeneration. Copyright

Spatial and temporal expression patterns of the epithelial cell adhesion molecule (EpCAM/EGP-2) in developing and adult kidneys.

Background: The epithelial cell adhesion molecule (EpCAM) is expressed by most epithelia and is involved in processes fundamental for morphogenesis, including cell-cell adhesion, proliferation, differentiation, and migration. Previously, a role for EpCAM in pancreatic morphogenesis was confirmed in vitro. Furthermore, changes in the EpCAM expression pattern were found in developing lung and thymus and in the regenerating liver. Therefore, EpCAM was proposed to be a morphoregulatory molecule. Methods: Using immunohistochemistry, the expression pattern of human and murine homologues of EpCAM was characterized in adult and embryonic kidneys from humans and human-EpCAM (hEpCAM)-transgenic mice. Results: EpCAM expression was found in the ureteric bud throughout nephrogenesis. EpCAM was not expressed in the metanephric mesenchyme. In comma- and S-shaped bodies, both metanephric mesenchyme derived structures, EpCAM expression appeared by E13.5. In adult kidneys, most epithelia expressed varying levels of EpCAM, as confirmed by double staining for human EpCAM and segment-specific nephron markers. Podocytes were EpCAM negative. At the cellular level, the EpCAM expression shifted from apical in embryonic to basolateral in adult kidneys. Conclusions: The spatiotemporal expression pattern of EpCAM changes during nephrogenesis. In the adult kidney, the expression varies markedly along the nephron. These data provide a basis for further studies on EpCAM in developing and adult kidneys.