Marcel H. J. Ruiters

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.

Induced DNA demethylation by targeting Ten-Eleven Translocation 2 to the human ICAM-1 promoter

Increasing evidence indicates that active DNA demethylation is involved in several processes in mammals, resulting in developmental stage-specificity and cell lineage-specificity. The recently discovered Ten-Eleven Translocation (TET) dioxygenases are accepted to be involved in DNA demethylation by initiating 5-mC oxidation. Aberrant DNA methylation profiles are associated with many diseases. For example in cancer, hypermethylation results in silencing of tumor suppressor genes. Such silenced genes can be re-expressed by epigenetic drugs, but this approach has genome-wide effects. In this study, fusions of designer DNA binding domains to TET dioxygenase family members (TET1, -2 or -3) were engineered to target epigenetically silenced genes (ICAM-1, EpCAM). The effects on targeted CpGs’ methylation and on expression levels of the target genes were assessed. The results indicated demethylation of targeted CpG sites in both promoters for targeted TET2 and to a lesser extent for TET1, but not for TET3. Interestingly, we observed re-activation of transcription of ICAM-1. Thus, our work suggests that we provided a mechanism to induce targeted DNA demethylation, which facilitates re-activation of expression of the target genes. Furthermore, this Epigenetic Editing approach is a powerful tool to investigate functions of epigenetic writers and erasers and to elucidate consequences of epigenetic marks.

Telomerase activity as a biomarker for (pre)neoplastic cervical disease in scrapings and frozen sections from patients with abnormal cervical smear.

PURPOSE To evaluate the diagnostic value of semi-quantitative telomerase activity assessment in cervical scrapings together with human papillomavirus (HPV) typing for detection of (pre)neoplastic cervical lesions and to compare telomerase activity in cervical scrapings and frozen specimens from the same patients. PATIENTS AND METHODS A cross-sectional study was performed in 161 patients referred for an abnormal cervical cytology report. In cervical scrapings, telomerase activity was determined by modified telomere repeat amplification protocol (TRAP) assay and HPV typing by polymerase chain reaction (PCR) with general and type-specific primers. Final diagnosis was made by pathologic examination of biopsy and/or loop excision specimens. RESULTS Telomerase activity was detectable in assessable scrapings from one of nine (11%) patients without cervical intraepitheleal neoplasia (CIN), in three of 26 (12%) with CIN I, eight of 35 (22%) with CIN II, 18 of 62 (29%) with CIN III, and four of 13 (31%) with cancer. Sensitivity and negative predictive value of the TRAP assay for CIN II/III and cancer lesions were 25% and 28%, respectively, while specificity for no CIN or CIN I was 89%. In representative frozen sections, frequency of detectable telomerase activity was related to grade of CIN/cancer; none of 21 normal cervices, none of two CIN I, two of 12 (17%) CIN II, 10 of 31 (32%) CIN III, and 18 of 21 (86%) cervical cancer lesions were telomerase-positive (P < .0005). Telomerase activity levels in paired scrapings and frozen sections appeared to be only weakly related; telomerase-positive sections with negative scrapings and vice versa (only in CIN III) were observed. In oncogenic HPV-negative scrapings (n = 14), no telomerase activity was detected, but in frozen sections, telomerase activity levels appeared to be unrelated to presence of specific HPV types. CONCLUSION Telomerase activity is more frequent in higher grade CIN/cervical cancer lesions. Telomerase activity assessment in cervical scrapings has a low sensitivity for CIN II/III and/or cervical cancer and does not appear to be useful in primary screening for cervical cancer. However, increased telomerase activity in frozen CIN sections may be a possible marker of progressive disease.

Detection of micrometastatic breast cancer by means of real time quantitative RT-PCR and immunostaining in perioperative blood samples and sentinel nodes

The aim of our study was to detect micrometastatic breast cancer by epithelial glycoprotein‐2 (EGP‐2) and cytokeratin 19 (CK19), using immunostaining and real time quantitative reverse transcriptase‐polymerase chain reaction (qRT‐PCR). Fifty‐eight breast cancer patients, 52 primary tumors, 75 sentinel nodes (SN) and 149 peripheral blood (PB) samples (from before, during and 4 days after operation) were examined. Immunostaining was performed with antibodies directed against EGP‐2 and CK19. Detection limits were one Michigan Cancer Foundation‐7 (MCF‐7) breast cancer cell line cell/2.106 leukocytes (immunostaining) and one MCF‐7 cell/106 leukocytes qRT‐PCR. Control noncancer lymph nodes (n = 10) showed nonspecific CK19 staining, but were qRT‐PCR negative; control healthy volunteer PB (n = 11) was always negative. Primary tumor samples, all positive with immunostaining, showed a wide variation of EGP‐2 (>104 fold) and CK19 mRNA expression (>103 fold). SN (n = 19) from 16 patients were tumor‐positive with routine haematoxylin‐eosin (H&E) and/or immunostaining. SN tumor presence was positively correlated to qRT‐PCR expression, but 3 tumor‐positive SN were false negative with qRT‐PCR. Three SN were qRT‐PCR positive, while tumor negative with H&E and/or immunostaining. No immunostaining positive PB was observed, but 19 patients (33%) had one or more qRT‐PCR positive PB samples. We concluded that primary tumors have varying expressions of EGP‐2 and CK19 mRNA. Both markers can be used in qRT‐PCR to obtain adequate sensitivity for single tumor cell detection. In SN, immunostaining appears more sensitive/specific than H&E or qRT‐PCR for tumor detection. No immunostaining positivity was found in PB, while 33% of patients had qRT‐PCR positive PB. The clinical value of these findings will have to be clarified.

Regulation of spontaneous and TNF/IFN-induced IL-6 expression in two human ovarian-carcinoma cell lines.

Autocrine and paracrine production of interleukin‐6 (IL‐6) is considered to be involved in the ongoing proliferation of ovarian‐cancer cells. In view of the variability in IL‐6 expression between various ovarian‐cancer cells, we questioned whether differences in IL‐6‐gene regulation might be observed in ovarian tumor cells with and without IL‐6 expression. The CAOV‐3 cell line spontaneously secreted IL‐6, which was enhanced by tumor necrosis factor‐α (877 ± 89 vs. 8,452 ± 1,762 pg/ml, x ± sd, p < 0.01). The electrophoretic mobility shift assay (EMSA) demonstrated that basic IL‐6 expression was associated with DNA binding of activator protein‐1 (AP‐1) and nuclear factor IL‐6 (NF‐IL6). Nuclear factor kappa‐B (NF‐κB), which consisted mainly of p65‐NF‐κB was induced in response to TNF‐α stimulation. A2780 cells did not express IL‐6, either spontaneously or after stimulation with TNF‐α. EMSAs, showed spontaneous AP‐1 but no NF‐IL6 or NF‐κB DNA binding. TNF‐α stimulation enhanced AP‐1 and induced NF‐κB but no NF‐IL6 DNA binding in these cells. NF‐IL6 protein, however, was detected in nuclear extracts of these cells by Western blotting. In contrast, IL‐6‐promoter transfection studies showed no difference in promoter activation between CAOV‐3 and A2780. This study reveals that differential IL‐6‐gene expression observed in ovarian‐cancer cell lines is independent of NF‐IL6 activation. Int. J. Cancer 82:244–249, 1999.

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.

Writing of H3K4Me3 overcomes epigenetic silencing in a sustained but context-dependent manner

Histone modifications reflect gene activity, but the relationship between cause and consequence of transcriptional control is heavily debated. Recent developments in rewriting local histone codes of endogenous genes elucidated instructiveness of certain marks in regulating gene expression. Maintenance of such repressive epigenome editing is controversial, while stable reactivation is still largely unexplored. Here we demonstrate sustained gene re-expression using two types of engineered DNA-binding domains fused to a H3K4 methyltransferase. Local induction of H3K4me3 is sufficient to allow re-expression of silenced target genes in various cell types. Maintenance of the re-expression is achieved, but strongly depends on the chromatin microenvironment (that is, DNA methylation status). We further identify H3K79me to be essential in allowing stable gene re-expression, confirming its role in epigenetic crosstalk for stable reactivation. Our approach uncovers potent epigenetic modifications to be directly written onto genomic loci to stably activate any given gene.

Targeted SAINT-O-Somes for improved intracellular delivery of siRNA and cytotoxic drugs into endothelial cells

In non-phagocytic cells such as endothelial cells, processing of liposomes and subsequent release of drug content is often inefficient due to the absence of professional processing machinery, which limits pharmacological efficacy. We therefore developed a liposome based drug delivery system with superior intracellular release characteristics. The design was based on long circulating conventional liposomes that were formulated with a cationic amphiphile, 1-methyl-4-(cis-9-dioleyl)methyl-pyridinium-chlorid (SAINT-C18). These so-called SAINT-O-Somes had a diameter of 100 nm, were as stable as conventionally formulated liposomes, and showed superior release of their content at pH conditions that liposomes encounter when they are endocytosed by cells. Attachment of anti-E-selectin specific antibodies to the distal end of surface grafted poly(ethylene glycol) resulted in immuno-SAINT-O-Somes that were as efficiently taken up by inflammation activated endothelial cells as conventional anti-E-selectin specific immunoliposomes. More importantly, intracellular release of calcein encapsulated in these targeted SAINT-O-Somes was 10 fold higher as compared to the release of calcein from conventional liposomes. For intracellular delivery siRNA into activated endothelial cells, formulation with SAINT-C18 was a necessity to induce a specific down-regulation of gene expression of VE-cadherin. Additionally, targeted doxorubicin loaded SAINT-O-Somes decreased endothelial cell viability significantly more than targeted conventional doxorubicin liposomes. SAINT-O-Somes therefore represent a new class of lipid based particles with superior drug release characteristics that can be applied for the efficacious intracellular delivery of hydrophilic drugs including siRNA.

Anti-VCAM-1 and anti-E-selectin SAINT-O-Somes for selective delivery of siRNA into inflammation-activated primary endothelial cells.

Activated endothelial cells play a pivotal role in the pathology of inflammatory diseases and present a rational target for therapeutic intervention by endothelial specific delivery of short interfering RNAs (siRNA). This study demonstrates the potential of the recently developed new generation of liposomes based on cationic amphiphile SAINT-C18 (1-methyl-4-(cis-9-dioleyl)methyl-pyridinium-chloride) for functional and selective delivery of siRNA into inflamed primary endothelial cells. To create specificity for inflamed endothelial cells, these so-called SAINT-O-Somes were harnessed with antibodies against vascular cell adhesion protein 1 (VCAM-1) or respectively E-selectin and tested in TNF-α activated primary endothelial cells from venous and aortic vascular beds. Both targeted SAINT-O-Somes carrying siRNA against the endothelial gene VE-cadherin specifically downregulated its target mRNA and protein without exerting cellular toxicity. SAINT-O-Somes formulated with siRNA formed small particles (106 nm) with a 71% siRNA encapsulation efficiency. SAINT-O-Somes were stable in the presence of serum at 37 °C, protected siRNA from degradation by serum RNases, and after i.v. injection displayed pharmacokinetic comparable to conventional long circulating liposomes. These anti-VCAM-1 and anti-E-selectin SAINT-O-Somes are thus a novel drug delivery system that can achieve specific and effective delivery of siRNA into inflamed primary endothelial cells and have physicochemical features that comply with in vivo application demands.

Targeted transfection increases siRNA uptake and gene silencing of primary endothelial cells in vitro--a quantitative study

Applications of small-interfering RNA (siRNA) call for specific and efficient delivery of siRNA into particular cell types. We developed a novel, non-viral targeting system to deliver siRNA specifically into inflammation-activated endothelial cells. This was achieved by conjugating the cationic amphiphilic lipid SAINT to antibodies recognizing the inflammatory cell adhesion molecule E-selectin. These anti-E-selectin-SAINT lipoplexes (SAINTarg) maintained antigen recognition capacity of the parental antibody in vitro, and ex vivo in human kidney tissue slices subjected to inflammatory conditions. Regular SAINT mediated transfection resulted in efficient gene silencing in human microvascular endothelial cells (HMEC-1) and conditionally immortalized glomerular endothelial cells (ciGEnC). However, primary human umbilical vein endothelial cells (HUVEC) transfected poorly, a phenomenon that we could quantitatively correlate with a cell-type specific capacity to facilitate siRNA uptake. Importantly, SAINTarg increased siRNA uptake and transfection specificity for activated endothelial cells. Transfection with SAINTarg delivered significantly more siRNA into activated HUVEC, compared to transfection with non-targeted SAINT. The enhanced uptake of siRNA was corroborated by improved silencing of both gene- and protein expression of VE-cadherin in activated HUVEC, indicating that SAINTarg delivered functionally active siRNA into endothelial cells. The obtained results demonstrate a successful design of a small nucleotide carrier system with improved and specific siRNA delivery into otherwise difficult-to-transfect primary endothelial cells, which in addition reduced considerably the amount of siRNA needed for gene silencing.