Sabine Hombach-Klonisch

Cancer stem cell markers in common cancers – therapeutic implications

Rapid advances in the cancer stem cell (CSC) field have provided cause for optimism for the development of more reliable cancer therapies in the future. Strategies aimed at efficient targeting of CSCs are becoming important for monitoring the progress of cancer therapy and for evaluating new therapeutic approaches. Here, we characterize and compare the specific markers that have been found to be present on stem cells, cancer cells and CSCs in selected tissues (colon, breast, liver, pancreas and prostate). We then discuss future directions of this intriguing new research field in the context of new diagnostic and therapeutic opportunities.

Adult stem cells and their trans-differentiation potential—perspectives and therapeutic applications

Stem cells are self-renewing multipotent progenitors with the broadest developmental potential in a given tissue at a given time. Normal stem cells in the adult organism are responsible for renewal and repair of aged or damaged tissue. Adult stem cells are present in virtually all tissues and during most stages of development. In this review, we introduce the reader to the basic information about the field. We describe selected stem cell isolation techniques and stem cell markers for various stem cell populations. These include makers for endothelial progenitor cells (CD146/MCAM/MUC18/S-endo-1, CD34, CD133/prominin, Tie-2, Flk1/KD/VEGFR2), hematopoietic stem cells (CD34, CD117/c-Kit, Sca1), mesenchymal stem cells (CD146/MCAM/MUC18/S-endo-1, STRO-1, Thy-1), neural stem cells (CD133/prominin, nestin, NCAM), mammary stem cells (CD24, CD29, Sca1), and intestinal stem cells (NCAM, CD34, Thy-1, CD117/c-Kit, Flt-3). Separate section provides a concise summary of recent clinical trials involving stem cells directed towards improvement of a damaged myocardium. In the last part of the review, we reflect on the field and on future developments.

A mouse model of human mucopolysaccharidosis IX exhibits osteoarthritis

Hyaluronidases are endoglycosidases that hydrolyze hyaluronan (HA), an abundant component of the extracellular matrix of vertebrate connective tissues. Six human hyaluronidase-related genes have been identified to date. Mutations in one of these genes cause a deficiency of hyaluronidase 1 (HYAL1) resulting in a lysosomal storage disorder, mucopolysaccharidosis (MPS) IX. We have characterized a mouse model of MPS IX and compared its phenotype with the human disease. The targeted Hyal1 allele in this model had a neomycin resistance cassette in exon 2 that replaced 753 bp of the coding region containing the predicted enzyme active site. As a result, Hyal1(-/-) animals had no detectable wild-type Hyal1 transcript, protein or serum activity. Hyal1 null animals were viable, fertile and showed no gross abnormalities at 1 year and 8 months of age. Histological studies of the knee joint showed a loss of proteoglycans occurring as early as 3 months that progressed with age. An increased number of chondrocytes displaying intense pericellular and/or cytoplasmic HA staining were detected in the epiphyseal and articular cartilage of null mice, demonstrating an accumulation of HA. Elevations of HA were not detected in the serum or non-skeletal tissues, indicating that osteoarthritis is the key disease feature in a Hyal1 deficiency. Hyal3 expression was elevated in Hyal1 null mice, suggesting that Hyal3 may compensate in HA degradation in non-skeletal tissues. Overall, the murine MPS IX model displays the key features of the human disease.

Cancer stem cells as targets for cancer therapy: selected cancers as examples

It is becoming increasingly evident that cancer constitutes a group of diseases involving altered stem-cell maturation/differentiation and the disturbance of regenerative processes. The observed malignant transformation is merely a symptom of normal differentiation processes gone astray rather than the primary event. This review focuses on the role of cancer stem cells (CSCs) in three common but also relatively under-investigated cancers: head and neck, ovarian, and testicular cancer. For didactic purpose, the physiology of stem cells is first introduced using hematopoietic and mesenchymal stem cells as examples. This is followed by a discussion of the (possible) role of CSCs in head and neck, ovarian, and testicular cancer. Aside from basic information about the pathophysiology of these cancers, current research results focused on the discovery of molecular markers specific to these cancers are also discussed. The last part of the review is largely dedicated to signaling pathways active within various normal and CSC types (e.g. Nanog, Nestin, Notch1, Notch2, Oct3 and 4, Wnt). Different elements of these pathways are also discussed in the context of therapeutic opportunities for the development of targeted therapies aimed at CSCs. Finally, alternative targeted anticancer therapies arising from recently identified molecules with cancer-(semi-)selective capabilities (e.g. apoptin, Brevinin-2R) are considered.

Differential expression and regulation of nuclear oligomerization domain proteins NOD1 and NOD2 in human endometrium: a potential role in innate immune protection and menstruation.

Nuclear oligomerization domains (NODs) are cytosolic pattern recognition receptors (PRRs), present in epithelial cells, monocytes and dendritic cells. This study details their expression, regulation and role in human endometrium. Real-time PCR showed that NOD1 mRNA is constitutively expressed in endometrium. NOD2 is up-regulated in the late secretory phase of the menstrual cycle suggesting a role in menstruation. Both proteins are immunolocalized in endometrial epithelium, stroma and endothelium. In first trimester, decidua NODs are present in decidualized stroma. NOD function was examined in endometrial stromal cells (ESCs) and endometrial epithelial cells (EEpCs) in vitro. IkappaBalpha is up-regulated by stimulation of ESC and EEpC with an NOD1 ligand. IkappaBalpha, IL-8 and TNFalpha mRNA expression is increased in EEpC by a NOD2 ligand. NOD2 mRNA expression increases in response to IL-1 treatment while NOD1 transcripts are unaltered. NOD1 mRNA is increased in an in vitro model of decidualization of ESC. In summary, we report expression of NOD1 and NOD2 in human endometrium and show that they are differentially regulated. NOD2 and, to a lesser extent, NOD1 can function to increase expression of innate immune molecules in endometrium. NODs may have a role in innate immune protection in the uterus and NOD2 may regulate inflammation associated with menstruation.

Murine Hyaluronidase 2 Deficiency Results in Extracellular Hyaluronan Accumulation and Severe Cardiopulmonary Dysfunction

Background: The role of hyaluronidase 2 in hyaluronan degradation and cardiopulmonary function is largely uncharacterized. Results: Hyaluronidase 2-deficient mice accumulate extracellular hyaluronan, leading to defective cardiopulmonary function. Conclusion: Hyaluronidase 2 is essential for hyaluronan degradation and normal heart and lung health. Significance: Our studies suggest that hyaluronidase 2 deficiency should be considered as a contributor to cardiac pathologies in humans. Hyaluronidase (HYAL) 2 is a membrane-anchored protein that is proposed to hydrolyze hyaluronan (HA) to smaller fragments that are internalized for breakdown. Initial studies of a Hyal2 knock-out (KO) mouse revealed a mild phenotype with high serum HA, supporting a role for HYAL2 in HA breakdown. We now describe a severe cardiac phenotype, deemed acute, in 54% of Hyal2 KO mice on an outbred background; Hyal2 KO mice without the severe cardiac phenotype were designated non-acute. Histological studies of the heart revealed that the valves of all Hyal2 KO mice were expanded and the extracellular matrix was disorganized. HA was detected throughout the expanded valves, and electron microscopy confirmed that the accumulating material, presumed to be HA, was extracellular. Both acute and non-acute Hyal2 KO mice also exhibited increased HA in the interstitial extracellular matrix of atrial cardiomyocytes compared with control mice. Consistent with the changes in heart structure, upper ventricular cardiomyocytes in acute Hyal2 KO mice demonstrated significant hypertrophy compared with non-acute KO and control mice. When the lungs were examined, evidence of severe fibrosis was detected in acute Hyal2 KO mice but not in non-acute Hyal2 KO or control mice. Total serum and heart HA levels, as well as size, were increased in acute and non-acute Hyal2 KO mice compared with control mice. These findings indicate that HYAL2 is essential for the breakdown of extracellular HA. In its absence, extracellular HA accumulates and, in some cases, can lead to cardiopulmonary dysfunction. Alterations in HYAL2 function should be considered as a potential contributor to cardiac pathologies in humans.

Expression and function of Toll-like receptors in human endometrial epithelial cell lines

In mammals, Toll-like receptors (TLRs) are the principal family of innate immune pattern recognition receptors (PRRs). The main function for TLRs is the detection of molecular patterns associated with invading pathogens. We investigated TLR expression and function in three established human endometrial epithelial cell lines, including hTERT-EEC, HEC-1B and Ishikawa cells, and clarified the application of these endometrial cell lines as in vitro models for studying TLR expression and function in the female reproductive tract. TLR gene expression was examined by RT-PCR and protein localization by immunohistochemistry. Our results showed that TLR expression in these cell lines is comparable to published literature on TLR expression in primary human endometrial tissue. TLR function was investigated by the detection of IL-6 and IL-8 production by ELISA in response to TLR2, TLR3, TLR5, TLR7 and TLR9 ligands. We found that hTERT-EEC cells were responsive to TLR5 ligand and HEC-1B cells respond to TLR3 and TLR5 ligands. In contrast, Ishikawa cells respond only to PMA/I which was used as a positive control for IL-8 production. Finally, we investigated the influence of flagellin as a TLR5 stimulant on TLR5 expression in these cell lines by QPCR. Our results showed that the endometrial cell lines showed a tendency for increased TLR5 expression in response to flagellin stimulation and in hTERT-EEC cells this tendency was statistically significant. These results suggest that hTERT-EEC, HEC-1B and Ishikawa cell lines can be used as in vitro models to investigate innate immune responses of endometrial cells in the female reproductive tract.

Relaxin reduces xenograft tumour growth of human MDA-MB-231 breast cancer cells

IntroductionRelaxin levels are increased in cases of human breast cancer and has been shown to promote cancer cell migration in carcinoma cells of the breast, prostate gland and thyroid gland. In oestrogen receptor alpha-negative MDA-MB-231 human breast cancer cells, relaxin was shown to down-regulate the metastasis-promoting protein S100A4 (metastasin), a highly significant prognostic factor for poor survival in breast cancer patients. The cellular mechanisms of relaxin exposure in breast cancer cells are not fully understood. The aim of this study was to investigate short-term and long-term effects of relaxin on cancer cell motility and S100A4 expression and to determine the long-term effects of relaxin on in vivo tumour growth in an oestrogen-independent context.MethodWe have established stable transfectants of highly invasive oestrogen-receptor alpha-negative MDA-MB-231 human breast cancer cells with constitutive expression of bioactive H2-relaxin (MDA/RLN2). RLN2 secretion was determined by ELISA. Relaxin receptor RXFP1 (Relaxin-family-peptide) was detected by reverse transcription (RT) PCR and its activation was assessed by induction of cyclic adenosine monophosphate (cAMP). Stable MDA/RLN2 clones and RLN2 treated MDA-MB-231 cells were subjected to motility and in vitro-invasion assays. Proliferation was assessed in bromodeoxyuridine (BrdU) and MTT assays. S100A4 expression was determined by RT-PCR and Western blot. Specific small interfering RNA was employed to down-regulate relaxin receptor and S100A4. MDA/EGFP vector control and two MDA/RLN2 clones were injected subcutaneously in nude mice to determine tumour growth and cancer cell invasiveness in vivo. Xenograft tumour tissues were assessed by histology and immunohistochemistry and frozen tissues were used for the detection of S100A4 and RLN2.ResultsShort-term exposure to relaxin for 24 hours increased cell motility in a relaxin receptor-dependent manner. This increase in cell motility was mediated by S100A4. Long-term exposure to relaxin secreted from stable transfectants reduced cell motility and in vitro invasiveness. Relaxin decreased cell proliferation and down-regulated cellular S100A4 levels in MDA-MB-231 and T47D breast cancer cells. Stable MDA/RLN2 transfectants produced smaller xenograft tumours containing reduced S100A4 protein levels in vivo.ConclusionOur results indicate that long-term exposure to relaxin confers growth inhibitory and anti-invasive properties in oestrogen-independent tumours in vivo, which may in part be mediated through a down-regulation of S100A4.

Relaxin-Like Ligand-Receptor Systems Are Autocrine/Paracrine Effectors in Tumor Cells and Modulate Cancer Progression and Tissue Invasiveness

Relaxin and INSL3 are novel autocrine/paracrine insulin-like hormones in tumor biology. Both effectors can bind to and activate the leucine-rich G-protein coupled receptors LGR7 relaxin receptor) or LGR8 (relaxin/INSL3 receptor). These relaxin-like ligand-receptor systems modulate cellular functions and activate signaling cascades in a tumor-specific context leading to changes in tumor cell proliferation, altered motility/migration and enhanced production/secretion ofpotent proteolytic enzymes. Matrix-metalloproteinases (MMP), tissue inhibitors of metalloproteinases (TIMP) and acid hydrolases such as cathepsins can facilitate tissue degradation and represent important proteolytic mediators of relaxin-like actions on tumor cell invasion and metastasis. This review presents recent new findings and emphasises the important functions of the relaxin/INSL3 ligand-receptor system as novel autocrine/paracrine effectors influencing tumor progression and tissue invasiveness.

INSL3 has tumor‐promoting activity in thyroid cancer

The functional role of INSL3 and its receptor RXFP2 in carcinogenesis is largely unknown. We have previously demonstrated (pro‐)cathepsin‐L as a target of INSL3 in human thyroid cancer cells facilitating penetration of tumor cells through elastin matrices. We demonstrate the expression of RXFP2 in human thyroid tissues and in mouse follicular thyroid epithelial cells using Cre‐recombinase transgene driven by Rxfp2 promoter. Recombinant and secreted INSL3 increased the motility of thyroid carcinoma (TC) cells in an autocrine/paracrine manner. This effect required the presence of RXFP2. We identified S100A4 as a novel INSL3 target molecule and showed that S100A4 facilitated INSL3‐induced enhanced motility. Stable transfectants of the human follicular TC cell line FTC‐133 expressing and secreting bioactive human INSL3 displayed enhanced anchorage‐independent growth in soft agar assays. Xenotransplant experiments in nude mice showed that INSL3, but not EGFP‐mock transfectants, developed fast‐growing and highly vascularized xenografts. We used human umbilical vein endothelial cells in capillary tube formation assays to demonstrate increased 2‐dimensional tube formations induced by recombinant human INSL3 and human S100A4 comparable to the effect of vascular endothelial growth factor used as positive control. We conclude that INSL3 is a powerful and multifunctional promoter of tumor growth and angiogenesis in human thyroid cancer cell xenografts. INSL3 actions involve RXFP2 activation and the secretion of S100A4 and (pro‐)cathepsin‐L.