Ruth Schwaninger

Bone morphogenetic protein 7 in the development and treatment of bone metastases from breast cancer.

Bone morphogenetic protein 7 (BMP7) counteracts the physiological epithelial-to-mesenchymal transition (EMT), a process that is indicative of epithelial plasticity. Because EMT is involved in cancer, we investigated whether BMP7 plays a role in breast cancer growth and metastasis. In this study, we show that decreased BMP7 expression in primary breast cancer is significantly associated with the formation of clinically overt bone metastases in patients with > or = 10 years of follow-up. In line with these clinical observations, BMP7 expression is inversely related to tumorigenicity and invasive behavior of human breast cancer cell lines. Moreover, BMP7 decreased the expression of vimentin, a mesenchymal marker associated with invasiveness and poor prognosis, in human MDA-MB-231 (MDA-231)-B/Luc(+) breast cancer cells under basal and transforming growth factor-beta (TGF-beta)-stimulated conditions. In addition, exogenous addition of BMP7 to TGF-beta-stimulated MDA-231 cells inhibited Smad-mediated TGF-beta signaling. Furthermore, in a well-established bone metastasis model using whole-body bioluminescent reporter imaging, stable overexpression of BMP7 in MDA-231 cells inhibited de novo formation and progression of osteolytic bone metastases and, hence, their metastatic capability. In line with these observations, daily i.v. administration of BMP7 (100 mug/kg/d) significantly inhibited orthotopic and intrabone growth of MDA-231-B/Luc(+) cells in nude mice. Our data suggest that decreased BMP7 expression during carcinogenesis in the human breast contributes to the acquisition of a bone metastatic phenotype. Because exogenous BMP7 can still counteract the breast cancer growth at the primary site and in bone, BMP7 may represent a novel therapeutic molecule for repression of local and bone metastatic growth of breast cancer.

Differential expression of TGFβ‐stimulated clone 22 in normal prostate and prostate cancer

The transforming growth factor‐β (TGFβ) superfamily and its downstream effector genes are key regulators of epithelial homeostasis. Altered expression of these genes may be associated with malignant transformation of the prostate gland. The cDNA array analysis of differential expression of the TGFβ superfamily and functionally related genes between patient‐matched noncancerous prostate (NP) and prostate cancer (PC) bulk tissue specimens highlighted two genes, namely TGFβ‐stimulated clone‐22 (TSC‐22) and Id4. Verification of their mRNA expression by real‐time PCR in patient‐matched NP and PC bulk tissue, in laser‐captured pure epithelial and cancer cells and in NP and PC cell lines confirmed TSC‐22 underexpression, but not Id4 overexpression, in PC and in human PC cell lines. Immunohistochemical analysis showed that TSC‐22 protein expression in NP is restricted to the basal cells and colocalizes with the basal cell marker cytokeratin 5. In contrast, all matched PC samples lack TSC‐22 immunoreactivity. Likewise, PC cell lines do not show detectable TSC‐22 protein expression as shown by immunoblotting. TSC‐22 should be considered as a novel basal cell marker, potentially useful for studying lineage determination within the epithelial compartment of the prostate. Conversely, lack of TSC‐22 seems to be a hallmark of malignant transformation of the prostate epithelium. Accordingly, TSC‐22 immunohistochemistry may prove to be a diagnostic tool for discriminating benign lesions from malignant ones of the prostate. The suggested tumour suppressor function of TSC‐22 warrants further investigation on its role in prostate carcinogenesis and on the TSC‐22 pathway as a candidate therapeutic target in PC.

The Role of the BMP Signaling Antagonist Noggin in the Development of Prostate Cancer Osteolytic Bone Metastasis

Members of the BMP and Wnt protein families play a relevant role in physiologic and pathologic bone turnover. Extracellular antagonists are crucial for the modulation of their activity. Lack of expression of the BMP antagonist noggin by osteoinductive, carcinoma-derived cell lines is a determinant of the osteoblast response induced by their bone metastases. In contrast, osteolytic, carcinoma-derived cell lines express noggin constitutively. We hypothesized that cancer cell-derived noggin may contribute to the pathogenesis of osteolytic bone metastasis of solid cancers by repressing bone formation. Intra-osseous xenografts of PC-3 prostate cancer cells induced osteolytic lesions characterized not only by enhanced osteoclast-mediated bone resorption, but also by decreased osteoblast-mediated bone formation. Therefore, in this model, uncoupling of the bone remodeling process contributes to osteolysis. Bone formation was preserved in the osteolytic lesions induced by noggin-silenced PC-3 cells, suggesting that cancer cell-derived noggin interferes with physiologic bone coupling. Furthermore, intra-osseous tumor growth of noggin-silenced PC-3 cells was limited, most probably as a result of the persisting osteoblast activity. This investigation provides new evidence for a model of osteolytic bone metastasis where constitutive secretion of noggin by cancer cells mediates inhibition of bone formation, thereby preventing repair of osteolytic lesions generated by an excess of osteoclast-mediated bone resorption. Therefore, noggin suppression may be a novel strategy for the treatment of osteolytic bone metastases.

The Molecular Signature of the Stroma Response in Prostate Cancer-Induced Osteoblastic Bone Metastasis Highlights Expansion of Hematopoietic and Prostate Epithelial Stem Cell Niches

The reciprocal interaction between cancer cells and the tissue-specific stroma is critical for primary and metastatic tumor growth progression. Prostate cancer cells colonize preferentially bone (osteotropism), where they alter the physiological balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption, and elicit prevalently an osteoblastic response (osteoinduction). The molecular cues provided by osteoblasts for the survival and growth of bone metastatic prostate cancer cells are largely unknown. We exploited the sufficient divergence between human and mouse RNA sequences together with redefinition of highly species-specific gene arrays by computer-aided and experimental exclusion of cross-hybridizing oligonucleotide probes. This strategy allowed the dissection of the stroma (mouse) from the cancer cell (human) transcriptome in bone metastasis xenograft models of human osteoinductive prostate cancer cells (VCaP and C4-2B). As a result, we generated the osteoblastic bone metastasis-associated stroma transcriptome (OB-BMST). Subtraction of genes shared by inflammation, wound healing and desmoplastic responses, and by the tissue type-independent stroma responses to a variety of non-osteotropic and osteotropic primary cancers generated a curated gene signature (“Core” OB-BMST) putatively representing the bone marrow/bone-specific stroma response to prostate cancer-induced, osteoblastic bone metastasis. The expression pattern of three representative Core OB-BMST genes (PTN, EPHA3 and FSCN1) seems to confirm the bone specificity of this response. A robust induction of genes involved in osteogenesis and angiogenesis dominates both the OB-BMST and Core OB-BMST. This translates in an amplification of hematopoietic and, remarkably, prostate epithelial stem cell niche components that may function as a self-reinforcing bone metastatic niche providing a growth support specific for osteoinductive prostate cancer cells. The induction of this combinatorial stem cell niche is a novel mechanism that may also explain cancer cell osteotropism and local interference with hematopoiesis (myelophthisis). Accordingly, these stem cell niche components may represent innovative therapeutic targets and/or serum biomarkers in osteoblastic bone metastasis.

Functional characterization of Fas ligand on tumor cells escaping active specific immunotherapy

Mice transgenic for the rat HER-2/neu oncogene (rNeu-TG) developed spontaneous breast tumors that can escape a rNeu-specific immune response induced by active specific immunotherapy (ASI). The ability of these escape tumors to grow appeared to be due to upregulation of the Fas ligand (Fas-L) molecule. In an effort to develop tools for the better elucidation of the role of Fas-L and other regulatory mechanisms in tumor escape, we established cell lines derived from escape tumors. These tumor cell lines retained MHC class I, rNeu and Fas-L expression in vitro and formed tumors in vaccinated mice. Tumor growth was accompanied by permanent Fas-L expression in vivo, both in vaccinated and control vaccinated mice, indicating that these cells have acquired constitutive Fas-L expression. Moreover, these cells induced target cell apoptosis in vitro. Thus, these cells represent a unique tool to elucidate the importance of Fas-L expressed by tumors that escaped efficient systemic immune responses. Cell Death and Differentiation (2001) 8, 687–695

Agents Used for Chemoprevention of Prostate Cancer May Influence PSA Secretion Independently of Cell Growth in the LNCaP model of Human Prostate Cancer Progression

The aim of this study was to evaluate the inhibitory growth effects of different potential chemopreventive agents in vitro and to determine their influence on PSA mRNA and protein expression with an established screening platform.

Virosomes as new carrier system for cancer vaccines

HER-2/neu, a tumor-associated antigen (TAAg), plays a critical role in oncogenesis of various tumor types, and its selective overexpression by malignant tumor cells makes it an ideal target for immunotherapy. A prerequisite for clinical vaccines is the construction of safe and highly immunogenic reagents able to generate efficient immune responses against TAAg. Previous protein vaccines, consisting of the extracellular domain of HER-2/neu (pNeuECD), were shown to elicit an immune response that did not provide protection from transplantable tumors expressing HER-2/neu. Here we showed that virosomes, which consist of reconstituted viral envelopes without viral genetic material, can act as a carrier and an adjuvant for a truncated protein pNeuECD . Mice vaccinated with pNeuECD either encapsulated in virosomes or bound to the virosomal membrane (Vir-pNeuECD), generated rNeu-specific humoral and cytotoxic immune responses. In addition, Vir-pNeuECD induced significant tumor rejection and additionally did not lead to delayed tumor formation when compared with free pNeuECD in complete Freund’s adjuvant. There was no difference between the virosomal constructs. Taken together these results suggest that virosomes, as clinically approved safe vaccines, can be used to elicit both humoral and cell-mediated responses against TAAg and induce tumor rejection. Our model is providing important preclinical data to design human vaccination trials for patients with tumors overexpressing HER-2/neu, either as a primary vaccination or as a boost in combination with other vaccines in a context of an adjuvant treatment plan.

Effects of seven different mutations in the pho1 gene on enzymatic activity, glycosylation and secretion of acid phosphatase in Schizosaccharomyces pombe

SummaryStructural gene mutants of the cell-surface glycoprotein acid phosphatase of Schizosaccharomyces pombe were analysed to define structural determinants that are responsible for enzymatic activity, N-glycosylation and secretion. All seven defined mutations cause a single amino acid substitution in the mature acid phosphatase protein and destroy the enzymatic activity. The mutational lesions are distributed throughout the pho1 gene. A ser to phe substitution at position 349 abolishes enzymatic activity only and does not affect glycosylation and secretion. Two mutations create a new N-glycosylation site by substitution of pro at position 56 by phe and ser, respectively. This new site is apparently used in the mutants. Their core-glycosylated acid phosphatase is slightly larger than that of the wild type. Overglycosylation seems not to affect secretion. Four different mutations (a gly to asp substitution at position 281 and ser to phe substitutions at positions 150, 271 and 277) cause intracellular accumulation of enzymatically inactive core-glycosylated acid phosphatase precursor. These mutational lesions apparently block transport of acid phosphatase from the endoplasmic reticulum to the Golgi apparatus.