Daniel Keppler

Cystatin M: A Novel Candidate Tumor Suppressor Gene for Breast Cancer

The contribution of pericellular proteolysis to tumor progression is well documented. To better understand protease biology and facilitate clinical translation, specific proteolytic systems need to be better defined. In particular, the precise role of endogenous protease inhibitors still needs to be deciphered. We reported previously that cystatin M, a potent endogenous inhibitor of lysosomal cysteine proteases, significantly suppressed in vitro cell proliferation, migration, and Matrigel invasion. Here, we show that scid mice orthotopically implanted with breast cancer cells expressing cystatin M show significantly delayed primary tumor growth and lower metastatic burden in the lungs and liver when compared with mice implanted with mock controls. The incidence of metastasis, however, appeared to be unaltered between the cystatin M group and the control group. Experimental metastasis assays suggest that cystatin M suppressed tumor cell proliferation at the secondary site. By using laser capture microdissection and quantitative reverse transcription-polymerase chain reaction, we found consistent expression of cystatin M in normal human breast epithelial cells, whereas expression was decreased by 86% in invasive ductal carcinoma (IDC) cells of stage I to IV patients. Complete loss of expression of cystatin M was observed in two of three IDCs from stage IV patients. Immunohistochemical studies confirmed that expression of cystatin M in IDCs was partially or completely lost. We propose cystatin M as a novel candidate tumor suppressor gene for breast cancer.

Cystatin M suppresses the malignant phenotype of human MDA-MB-435S cells.

Proteases are involved in many aspects of tumor progression, including cell survival and proliferation, escape from immune surveillance, cell adhesion and migration, remodeling and invasion of the extracellular matrix. Several lysosomal cysteine proteases have been cloned and shown to be overexpressed in cancer; yet, despite the great potential for development of novel therapeutics, we still know little about the regulation of their proteolytic activity. Cystatins such as cystatin M are potent endogenous protein inhibitors of lysosomal cysteine proteases. Cystatin M is expressed in normal and premalignant human epithelial cells, but not in many cancer cell lines. Here, we examined the effects of cystatin M expression on malignant properties of human breast carcinoma MDA-MB-435S cells. Cystatin M was found to significantly reduce in vitro: cell proliferation, migration, Matrigel invasion, and adhesion to endothelial cells. Reduction of cell proliferation and adhesion to an endothelial cell monolayer were both independent of the inhibition of lysosomal cysteine proteases. In contrast, cell migration and matrix invasion seemed to rely on lysosomal cysteine proteases, as both recombinant cystatin M and E64 were able to block these processes. This study provides the first evidence that cystatin M may play important roles in safeguarding against human breast cancer.

Analysis of a truncated form of cathepsin H in human prostate tumor cells

Increased expression of proteases has been correlated with the malignant progression of a variety of tumors. We found a significant increase in cathepsin H expression in high-grade prostatic intraepithelial neoplasia and carcinoma of the prostate. Two forms of cathepsin H, the full-length form (CTSH) and a truncated form with a 12-amino acid deletion in its signal peptide region (CTSHΔ10–21), were identified by cDNA sequence analysis. This deletion occurred not at the genomic level but likely at the RNA processing level. Both forms are expressed in prostate tissues as well as LNCaP, PC-3, and DU-145 prostate cancer cell lines. The deletion within the signal peptide region affected the trafficking of cathepsin H. Fluorescence microscopy, subcellular fractionation, and activity data indicated that the truncated form was perinuclear and secreted and had a reduced lysosomal association as compared with the full-length cathepsin H. Furthermore, the truncated cathepsin H was enzymatically active. Therefore, an increase in overall cathepsin H expression, particularly in the truncated form with a high secretion propensity, may affect cell biological behaviors such as those associated with tumor progression.

CATHEPSIN B : MULTIPLE ENZYME FORMS FROM A SINGLE GENE AND THEIR RELATION TO CANCER

Overexpression and altered trafficking of cathepsin B characterize the malignant phenotype of tumor cells. Human cathepsin B is encoded by a single-copy gene located on chromosome 8p22. With its 13 exons, the gene encompasses at least 27 kb of DNA. Expression of cathepsin B can be regulated at transcriptional and posttranscriptional levels. Multiple cathepsin B mRNA species arising from alternative splicing may be related to tissue- and tumor-specific differences in expression. There is selective overexpression, increased activity, membrane association and secretion of cathepsin B in many etiologically different cancers. This suggests that cathepsin B may play a functional role in malignant progression. Recent clinical studies provide confirmatory evidence in that cathepsin B expression is a prognostic indicator in colon carcinoma.

Inactivation of the Cystatin E/M Tumor Suppressor Gene in Cervical Cancer

We have previously localized a cervical cancer tumor suppressor gene to a 300 kb interval of 11q13. Analysis of candidate genes revealed loss of expression of cystatin E/M, a lysosomal cysteine protease inhibitor, in 6 cervical cancer cell lines and 9 of 11 primary cervical tumors. Examination of the three exons in four cervical cancer cell lines, 19 primary tumors, and 21 normal controls revealed homozygous deletion of exon 1 sequences in one tumor. Point mutations were observed in six other tumors. Two tumors contained mutations at the consensus binding sites for cathepsin L, a lysosomal protease overexpressed in cervical cancer. Introduction of these two point mutations using site directed mutagenesis resulted in reduced binding of mutated cystatin E/M to cathepsin L. Although mutations were not observed in any cell lines, four cell lines and 12 of 18 tumors contained promoter hypermethylation. Reexpression of cystatin E/M was observed after 5′aza 2‐deoxycytidiene and/or Trichostatin A treatment of cervical cancer cell lines, HeLa and SiHa, confirming promoter hypermethylation. Ectopic expression of cystatin E/M in these two cell lines resulted in growth suppression. There was also suppression of soft agar colony formation by HeLa cells expressing the cystatin E/M gene. Reexpression of cystatin E/M resulted in decreased intracellular and extracellular expression of cathepsin L. Overexpression of cathepsin L resulted in increased cell growth which was inhibited by the reintroduction of cystatin E/M. We conclude, therefore, that cystatin E/M is a cervical cancer suppressor gene and that the gene is inactivated by somatic mutations and promoter hypermethylation.

Increased expression of mature cathepsin B in aging rat liver.

Abstract. Senescence has been proposed as an important safeguard against neoplasia. One of the hallmarks of cellular senescence in vitro as well as human aging in vivo is a reduced intracellular protein catabolism. The pathways affected and the mechanisms responsible for the decrease in overall protein turnover in aging cells are not well understood. Our aim was to determine whether or not expression of one of the major hepatic lysosomal cysteine peptidases, cathepsin B, changes during aging of Sprague-Dawley rats. Cathepsin B activity was assessed in whole rat liver homogenates, and was found to be increased fourfold (P≤0.001) in aged livers compared with younger counterparts. This was paralleled by an at least a twofold increase in mature cathepsin B protein. Nonetheless, Northern blot analysis of total liver RNA revealed no change in steady-state levels of cathepsin B mRNAs. These findings seem to contradict the present dogma according to which aging tissues have a reduced intracellular capacity to catabolise proteins. We propose that our earlier observation of the accumulation of T-kininogen, a potent but reversible cysteine peptidase inhibitor, in aging rat liver may provide a plausible explanation for this discrepancy.

Cathespin H Is an Fgf10 Target Involved in Bmp4 Degradation during Lung Branching Morphogenesis

During lung development, signaling by Fgf10 (fibroblast growth factor 10) and its receptor Fgfr2b is critical for induction of a gene network that controls proliferation, differentiation, and branching of the epithelial tubules. The downstream events triggered by Fgf10-Fgfr2b signaling during this process are still poorly understood. In a global screen for transcriptional targets of Fgf10, we identified Ctsh (cathepsin H), a gene encoding a lysosomal cysteine protease of the papain family, highly up-regulated in the developing lung epithelium. Here we show that among other cathepsin genes present in the lung, Ctsh is the only family member selectively induced by Fgf10 in the lung epithelium. We provide evidence that, during branching morphogenesis, epithelial expression of Ctsh overlaps temporally and spatially with that of Bmp4 (bone morphogenetic protein 4), another target of Fgf10. Moreover, we show that Ctsh controls the availability of mature Bmp4 protein in the embryonic lung and that inhibiting Ctsh activity leads to a marked accumulation of Bmp4 protein and disruption of branching morphogenesis. Tightly controlled levels of Bmp4 signaling are critical for patterning of the distal lung epithelium. Our study suggests a potentially novel posttranscriptional mechanism in which Ctsh rapidly removes Bmp4 from forming buds to limit Bmp4 action. The presence of both Ctsh and Bmp4 or Bmp4 signaling activity in other developing structures, such as the kidney, yolk sac, and choroid plexus, suggests a possible general role of Ctsh in regulating Bmp4 proteolysis in different morphogenetic events.

Role of cystatins in tumor neovascularization

Cystatins form a large superfamily of proteins with diverse biologic activities. All members of the cystatin superfamily share the presence of one, two or three cystatin domains. Cystatins were initially believed to act mainly as inhibitors of lysosomal cysteine proteases. In recent years, however, there has been increased awareness of additional or alternate biologic functions for these proteins. In this review, the authors will discuss the most recent findings and hypotheses that suggest that some members of the cystatin superfamily may play important roles during tumor progression. Special emphasis is given to their potential role as novel anti-angiogenic agents.

Inhibitors of Papain-Like Cysteine Peptidases in Cancer

Since the discovery of a polypeptide in chicken egg white (cew cystatin) that is able to inhibit the plant cysteine peptidase papain, investigators have identified numerous mammalian cysteine peptidases and cloned more than 40 cystatin genes. The cloning frenzy will probably continue for a few more years, raising classification and nomenclature problems in the papain- and cystatin super families. We now know that cysteine peptidases are involved in many biological processes, such as growth, differentiation, immunity, virulence, and death. Deregulation of these processes leads to tumor formation and metastasis. The present challenge is to define when and where each gene product is expressed and against what target(s) it is directed. The diversity of cancers and their intrinsic heterogeneity renders this task very difficult. In this review, we summarize what is presently known about cystatins, with particular emphasis on their function as inhibitors of lysosomal/endosomal cysteine peptidases. However, there is also evidence for other functions of cystatins. Is there a relationship between inhibitor function and these other functions? Why are there cytosolic and secreted inhibitors, when the target peptidases are vesicular? These are only a few questions that may be answered during the next 10 years.

Retroviral Expression of Human Cystatin Genes in HeLa Cells

Retroviral gene transfer is a highly efficient and effective method of stably introducing genetic material into the genome of specific cell types. The process involves the transfection of retroviral expression vectors into a packaging cell line, the isolation of viral particles, and the infection of target cell lines. Compared to traditional gene transfer methods such as liposome-mediated transfection, retroviral gene transfer allows for stable gene expression in cell populations without the need for lengthy selection and cloning procedures. This is particularly helpful when studying gene products that have negative effect on cell growth and viability. Here, we describe the retroviral transfer of cystatin cDNAs using HEK293-derived Phoenix packaging cells and human HeLa cervical carcinoma cells as target cells.