Michael Zepp

Sequential biphasic changes in claudin1 and claudin4 expression are correlated to colorectal cancer progression and liver metastasis

Terminal progression of colorectal cancer (CRC) culminates in liver metastasis. To identify genes that are involved in the metastatic phenotype, cDNA microarrays were used to analyse mRNA expression profiles of colorectal carcinoma (CC)531 rat colon adenocarcinoma cells for changes related to their homing into the liver. Briefly, CC531 cells were intraportally implanted into the liver of Wag‐Rij rats and re‐isolated after 3, 6, 9, 14 and 21 days. Compared to control CC531 cells, claudin1 and claudin4 were among the ≥8‐fold initially down‐regulated genes. The co‐culture of tumour cells with isolated rat hepatocytes and Kupffer cells did not induce down‐regulation of either claudin1 or 4. When the environment effective on circulating tumour cells was simulated by cell culture conditions favouring their adhesion, only claudin4 showed augmented expression. Knockdown of claudin1 and claudin4 mediated by small interfering RNA caused significantly increased migration and decreased clonogenic growth of tumour cells (P < 0.05), but had no effect on their proliferation. These experimental results were paralleled by increased claudin1 and claudin4 expression in human CRC samples in Union for International Cancer Control (UICC) stages I–III, as evaluated by real‐time PCR. Increased claudin4 levels were correlated with significantly reduced overall survival (log‐rank test, P= 0.018). Further, significantly (P < 0.05) reduced expression of claudin1 and claudin4 was observed in stage IV and liver metastasis by immunohistochemistry. In conclusion, sequential biphasic changes in claudin1 and claudin4 expression occur during the homing of rat CC531 CRC cells to the liver. This modulation is reflected by significant changes in claudin expression in human primary and metastatic CRC.

Biodistribution of antisense nanoparticles in mammary carcinoma rat model.

Efficient and specific delivery of antisenses (ASs) and protection of the sequences from degradation are critical factors for effective therapy. Sustained release nanoparticles (NP) offer increased resistance to nuclease degradation, increased amounts of AS uptake, and the possibility of control in dosing and sustained duration of AS administration. The biodegradable and biocompatible poly(D,L-lactic-co-glycolic acid) copolymer (PLGA) was utilized to encapsulate AS directed against osteopontin (OPN), which is a promising therapeutic target in mammary carcinoma. Whole body biodistribution of OPN AS NP was evaluated in comparison to naked AS, in intact and mammary carcinoma metastasis model bearing rats. Naked and NP encapsulated AS exhibited different biodistribution profiles. AS NP, in contrast to naked AS, tended to accumulate mostly in the spleen, liver, and at the tumor inoculation site. Drug levels in intact organs were negligible. The elimination of naked AS was faster, due to rapid degradation of the unprotected sequence. It is concluded that AS NP protect the AS from degradation, provide efficient AS delivery to the tumor tissue, and minimize AS accumulation in intact organs due to the AS sustained release profile as well as the favorable NP physicochemical properties.

Treatment of Breast Cancer Lytic Skeletal Metastasis Using a Model in Nude Rats

Cancer is a life-threatening disease, not as a result of the primary tumor that can be removed surgically in the vast majority of cases but from its metastatic spread to distant parts of the body. These metastases are often seen as a hopeless end-stage of the cancer disease and at this time only palliative treatments are applied. Some of the most prevalent solid tumors, such as breast-, lungand prostate cancers, metastasize into the skeleton and cause either osteolytic (destructive) or osteoblastic lesions. Both types are often accompanied by bone pain and increased bone fragility and thus are reason for extended suffering. In breast cancer, bone is the site of first distant relapse and the clinical course of these women is relatively long, with a median survival of 2-3 years (1, 2). Lytic skeletal metastases are present in over 90% of patients who die from breast cancer (3). Many factors are involved in the pathogenesis of lytic skeletal lesions among which the proteins osteopontin (OPN) and bone sialoprotein II (BSPII) are considered to play an important role. In patients with primary breast cancer, elevated serum BSPII was recognized as prognostic marker of subsequent bone metastasis and was associated with poor survival (4-8). BSPII is a noncollagenous protein of the extracellular bone matrix and a member of the SIBLING (Small Integrin-Binding Ligand, N-linked Glycoprotein) family. The SIBLINGs are mainly clustered on human chromosome 4, and include bone sialoprotein II, osteopontin, dentin matrix protein 1 (DMP1), matrix extracellular phosphoglycoprotein (MEPE) and dentin sialophosphoprotein (DSPP) (12). These proteins are normally expressed in mineralizing tissues of bone and teeth but are also found in different cancers (13). In normal bone, BSPII is expressed by osteoblasts, osteoclasts and other skeleton-associated cell types, especially at sites of new mineral formation (12, 14-16). In this case, BSPII is a potential nucleator of hydroxyapatite formation and a specific marker of osteoblast differentiation (14). The sialoprotein is involved in hydroxyapatite and collagen binding, as well as in the attachment of bone cells including fibroblasts, osteoblasts and osteoclasts to solid surfaces,

IDK1 is a rat monoclonal antibody against hypoglycosylated bone sialoprotein with application as biomarker and therapeutic agent in breast cancer skeletal metastasis

Changes in glycosylation are salient features of cancer cells. Here, we report on the diagnostic and therapeutic properties of IDK1, an antibody against tumour associated, hypoglycosylated bone sialoprotein (hypo‐BSP). The affinity of the rat monoclonal antibody IDK1 for hypo‐BSP, as determined by microscale thermophoresis, was three orders of magnitude higher than for mature BSP, whereas the mouse monoclonal antibody used had similar affinity for both BSP forms. IDK1 showed no activity against the proliferation or migration of normal or cancer cells growing in vitro. In vivo, however, IDK1 caused dose‐dependent regression of soft tissue and skeletal lesions in nude rats harbouring human MDA‐MB‐231 cells. At optimal dose, 80% of the treated rats showed complete remission of all tumour lesions. Analysis of BSP expression in vitro by fluorescence‐activated cell sorting (FACS) and immunocytochemistry showed basal levels of this protein, which were visible only in a fraction of these cells. Cells of the metastatic cell lines MDA‐MB‐231 and PC‐3 were more often positive for hypo‐BSP. In addition, there was co‐expression of both forms in some cells, but almost no co‐localization; rather, hypo‐BSP was present in the nucleus, and mature BSP was detected extra‐cellularly. Normal osteoblasts and osteoclasts were negative for hypo‐BSP. Breast cancer tissue, however, showed strong expression of mature BSP, which was present intra‐cellularly as well as in vesicles outside cells. Hypo‐BSP was present mainly in lesions from skeletal sites, thus explaining the antineoplastic activity of IDK1, which was high in lesions growing in the vicinity of the skeleton but low in lesions growing subcutaneously. Finally, hypo‐BSP was detected in specimens from breast cancer patients, with a significantly greater intensity in skeletal metastases as compared to the respective primary cancers. In conclusion, IDK‐1 is an antibody with diagnostic and therapeutic applications in skeletal metastases of breast cancer.

Upregulation of cell cycle genes in head and neck cancer patients may be antagonized by erufosine’s down regulation of cell cycle processes in OSCC cells

The TCGA database was analyzed to identify deregulation of cell cycle genes across 24 cancer types and ensuing effects on patient survival. Pan-cancer analysis showed that head and neck squamous cell carcinoma (HNSCC) ranks amongst the top four cancers showing deregulated cell cycle genes. Also, the median gene expression of all CDKs and cyclins in HNSCC patient samples was higher than that of the global gene expression. This was verified by IHC staining of CCND1 from HNSCC patients. When evaluating the quartiles with highest and lowest expression, increased CCND1/CDK6 levels had negative implication on patient survival. In search for a drug, which may antagonize this tumor profile, the potential of the alkylphosphocholine erufosine was evaluated against cell lines of the HNSCC subtype, oral squamous cell carcinoma (OSCC) using in-vitro and in-vivo assays. Erufosine inhibited growth of OSCC cell lines concentration dependently. Initial microarray findings revealed that cyclins and CDKs were down-regulated concentration dependently upon exposure to erufosine and participated in negative enrichment of cell cycle processes. These findings, indicating a pan-cdk/cyclin inhibition by erufosine, were verified at both, mRNA and protein levels. Erufosine caused a G2/M block and inhibition of colony formation. Significant tumor growth retardation was seen upon treatment with erufosine in a xenograft model. For the decreased cyclin D1 and CDK 4/6 levels found in tumor tissue, these proteins can serve as biomarker for erufosine intervention. The findings demonstrate the potential of erufosine as cell cycle inhibitor in HNSCC treatment, alone or in combination with current therapeutic agents.

Abstract P2-04-23: A monoclonal antibody against hypo-glycosylated bone sialoprotein II has application for diagnostic purposes in samples of breast cancer patients and for treatment of skeletal metastasis caused by MDA-MB-231 breast cancer cells in rats

The SIBLING protein bone sialoprotein II (BSP) has been implicated in lytic skeletal metastasis as it is expressed in a subset of primary breast cancers and can be detected at elevated levels in the serum of patients with increased risk to develop skeletal metastasis. The aim of this study was to investigate the potential application of a rat monoclonal antibody against hypo-glycosylated BSP (IDK1) for diagnostic and therapeutic purposes. The diagnostic part of this study was based on breast cancer specimens from the biobank / repository of the Institute of Pathology of the Municipal Hospital Kassel, Germany. Immune-histochemical analyses were performed with IDK1 for comparing BSP expression between ten human primary breast tumor sections and their corresponding bone metastatic tissue samples. The therapeutic part of this study was based on a model in nude rats, in which the rats were implanted with human MDA-MB-231 breast cancer cells for selective and orthotopic appearance of osteolytic skeletal lesions. Tumor bearing rats were treated with IDK1 starting at two or four weeks after tumor cell inoculation into the femoral artery of one hind leg. Tumor growth was monitored by light emission, caused by luciferase mediated metabolism of luciferin. Photon emission was recorded at regular intervals by a Xenogen IVIS 100 imaging system. After sacrifice, samples of lesions and apparently healthy tissues were investigated by HE tumor take rate 95%). In contrast, rats treated with the anti-BSP antibody did not show a significant increase in light emission nor a clinical deterioration. In fact, 8 of 10 rats receiving the antibody at a dose of 10 mg/kg/week starting at two weeks after tumor implantation did not show any light emission after 4 to 6 weeks (p = 0.01 versus control) as well as 6 of 10 rats receiving the antibody at the same dose starting at four weeks after tumor implantation (p In conclusion, the rat monoclonal antibody directed against BSP is a powerful tool with potential for diagnostic and therapeutic applications in breast cancer skeletal metastasis and warrants further development. Citation Format: Berger MR, Zepp M, Westphal G, Berger I, Armbruster FP. A monoclonal antibody against hypo-glycosylated bone sialoprotein II has application for diagnostic purposes in samples of breast cancer patients and for treatment of skeletal metastasis caused by MDA-MB-231 breast cancer cells in rats [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-04-23.

Abstract 2474: A rat monoclonal antibody against bone sialoprotein II shows differential activity in MDA-MB-231 cells growing in vitro or in vivo

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Elevated serum levels of the small integrin binding ligand N-linked glycoprotein (SIBLING) family member bone sialoprotein II (BSP) have been related to breast cancer skeletal metastasis. Also, knockdown of BSP was associated with significant anti-proliferative, anti-migratory and anti-clonogenic effects in MDA-MB-231 human breast cancer cells, and rats presenting with osteolytic metastases following femoral artery injection of these cells underwent complete remission following BSP knockdown, thus validating BSP as target. In order to follow a translational perspective, we used the IDK-1 rat monoclonal antibody against BSP (Immundiagnostik, Bensheim, Germany) for treating MDA-MB-231 cells in vitro and in vivo. MDA-MB-231 cells were exposed to IDK-1 for assessing its effect on proliferation and migration. The presence of BSP in the cytosol and on cell membranes was checked by FACS analysis. In addition, the location of BSP was investigated by immunocytochemistry. For in vivo experiments, 1×105 MDA-MB-231luc breast cancer cells were injected into the femoral artery of male nude rats with skeletal lesions developing subsequently in the respective hind leg. A preventive arm based on pretreated rats and / or MDA-MB-231 cells was compared with a treatment arm, in which antibody administration (10 mg/kg/week) started when tumor bearing rats had shown stable tumor growth. The appearance and growth of soft tissue tumors was monitored by luciferin induced light emission and recorded by a Xenogen IVIS 100 imaging system. Concomitant skeletal lesions were detected by CT scans. Tumors as well as skeletal lesions were subjected to pathohistological evaluation by hematoxylin and eosin staining as well as immunohistochemical staining for BSP. There was no effect of the anti-BSP antibody IDK-1 on the proliferation or migration of MDA-MB-231 cells. In line with this, FACS analysis revealed only low concentrations of BSP in MDA-MB-231 cells growing in vitro. Immunocytochemical staining for BSP showed that this SIBLING protein could be detected only in a minority of the MDA-MB-231 cells. However, when treating nude rats bearing fully established MDA-MB-231 tumors, administration of the IDK-1 antibody caused complete remissions in 80% of treated rats (10mg/kg/week for 6 weeks). When pre-treating the nude rats, however, and / or pre-exposing the MDA-MB-231 cells to IDK-1 in addition to treating the established tumors, the above mentioned effect could not be increased. Interestingly, histological evaluation of serial sections of MDA-MB-231 tumors growing in nude rats showed a very robust expression and vesicular secretion of BSP. It is concluded, that the in vivo growth of MDA-MB-231 cells is associated with dramatically increased expression and secretion of BSP, which then is a valid target for the anti-BSP antibody, leading to complete remissions of MDA-MB-231 tumors in nude rats. Citation Format: Michael Zepp, Irina Berger, Heidegard Hilbig, Franz-Paul Armbruster, Martin R. Berger. A rat monoclonal antibody against bone sialoprotein II shows differential activity in MDA-MB-231 cells growing in vitro or in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2474. doi:10.1158/1538-7445.AM2015-2474

Abstract 2614: Bone sialoprotein is an essential target in breast cancer skeletal metastasis

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Breast cancer is a leading cause of cancer related death in women due to onset of metastasis. Bone metastasis is the most frequent complication occurring in patients with advanced breast cancer and bone sialoprotein (BSP) is related to this process. However, the underlying mechanisms are not clear yet. Therefore, the aim of the study was to analyze BSP functions in greater detail and to decipher its signaling pathways contributing to bone metastasis. To that purpose a combination of the tetracycline-controlled transcription activation system (“Tet-Off system”) and RNA interference was used to initiate and maintain the conditional knockdown of BSP for any intended period. This new technique was established in MDA-MB-231 subclones by recombinase-mediated cassette exchange. Additionally, the cell clones were equipped with the reporter genes mCherry and firefly luciferase for testing their regulative properties and following their fate. In absence of doxycycline, the expression of a miRNA targeting BSP was activated and after six days of BSP knockdown the ensuing cellular, metastatic or molecular properties were monitored by fluorescent microscopy, flow cytometry analysis, assays for proliferation, migration and colony formation, as well as expression profiling analysis. Furthermore, the cell clones were examined in a nude rat model for soft tissue and osteolytic lesions after 2 to 6 weeks of miRNA treatment. The clones revealed good regulative properties to doxycycline. Phenotypic changes indicating apoptosis were observed after 6 days of conditional knockdown which was characterized by up to 86% decreased BSP levels and resulted in significant anti-proliferative, anti-migratory and anti-clonogenic effects in vitro. Additionally, the effect of miRNA-mediated BSP knockdown was assessed in vivo. Significant decreases (p < 0.03) and even complete remissions of soft tissue and osteolytic lesions were found following 3 and 6 weeks of miRNA treatment by bioluminescence and magneting resonance imaging, as well as volume computed tomography. The microarray data showed modulated expression in 1.3% of all genes, thus hinting to specific effects in response to BSP knockdown. These genes included increased expression of endoplasmic reticulum stress and apoptosis related genes (ATF3, CHOP), of transcription factor c-FOS, of the gene related to breast epithelial differentiation (ID2) and the tumor suppressor gene EGR1. Conversely, there was suppression of metastasis associated genes (CD44, IL11). These findings were confirmed by western blot for induction of intrinsic and extrinsic apoptotic pathways as shown by cleavage of caspases 8, 9, 3 and 7, and of PARP, as well as the upregulation of ATF3, DDIT3 (CHOP), c-FOS, ID2 and CD44. In conclusion, the role of BSP in the development of skeletal metastasis has been defined more precisely and renders this protein an attractive target in the treatment of this disease. Citation Format: Marineta Kovacheva, Michael Zepp, Stefan Berger, Martin R. Berger. Bone sialoprotein is an essential target in breast cancer skeletal metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2614. doi:10.1158/1538-7445.AM2014-2614