Metastasis Suppressor Protein Research Articles

Nm23 expression in retinoblastoma

Purpose: Nm23 is a metastasis-suppressor protein. Decreased nm23 protein contributes to aggressiveness in many tumors. Nm23 immunoreactivity was studied in retinoblastoma and correlated with differentiation and invasiveness. Methods: Immunohistochemistry was performed on 73 formalin-fixed, paraffin-embedded specimens of retinoblastoma. Western blot was conducted to confirm the immunohistochemical study. Prognostic features such as differentiation, invasion of choroid, optic nerve, and orbit, and metastasis were analyzed. Results: Intense nm23 immunoreactivity was seen in 61% of the retinoblastomas with no invasion and faint nm23 immunoreactivity was seen in 85% of the retinoblastomas with invasion (p < 0.001). Poorly differentiated retinoblastoma showed decreased nm23 immunoreactivity compared to well-differentiated retinoblastomas (p = 0.02). An inverse correlation was observed between invasion of choroid, optic nerve, orbit, and metastasis, and nm23 immunoreactivity. Western blot assays of fresh tumor extracts confirmed the immunohistochemical findings. Conclusions: Decreased nm23 immunoreactivity was seen in poorly differentiated retinoblastomas and in retinoblastomas with invasiveness. These findings may lay the groundwork for further studies to better understand the molecular mechanisms and provide a more accurate prediction of invasion and metastasis of retinoblastoma.

MIM-B, a putative metastasis suppressor protein, binds to actin and to protein tyrosine phosphatase delta.

We have found that MIM-B, a putative metastasis suppressor protein, is implicated in actin cytoskeletal control and interaction with a protein tyrosine phosphatase (PTP). MIM was originally described as a protein whose mRNA was Missing in Metastasis, as it was found not to be present in metastatic bladder carcinoma cell lines [Lee, Y. G., Macoska, J. A., Korenchuk, S. and Pienta, K. J. (2002) Neoplasia 4, 291-294]. We further characterized a variant of MIM, which we call MIM-B, and which we believe may be a link between tyrosine kinase signalling and the actin cytoskeleton. We have shown, using purified proteins and cell extracts, that MIM-B is an actin-binding protein, probably via a WASP (Wiskott-Aldrich syndrome protein)-homology 2 domain at its C-terminus. We have also found that MIM-B binds to the cytoplasmic domain of receptor PTPdelta. Expression of full-length MIM-B induces actin-rich protrusions resembling microspikes and lamellipodia at the plasma membrane and promotes disassembly of actin stress fibres. The C-terminal portion of MIM-B is localized in the cytoplasm and does not affect the actin cytoskeleton when expressed, while the N-terminal portion localizes to internal vesicles and probably targets the protein to membranes. We postulate that MIM-B may be a regulator of actin assembly downstream of tyrosine kinase signalling and that this activity may explain the involvement of MIM in the metastasis of cancer cells.

KAI1 Metastasis Suppressor Protein in Cervical Cancer

KAI1 Metastasis Suppressor Protein in Cervical Cancer

Downregulation of KAI1 Metastasis Suppressor Protein Is Associated with a Dismal Prognosis in Epithelial Ovarian Cancer

Objectives. The aim of this study was to investigate the impact of downregulation of KAI1 metastasis suppressor protein in epithelial ovarian cancer. In addition, correlation of KAI1 and p53 immunostaining was investigated.Methods. Expression of KAI1 and p53 was immunohistochemically determined in 107 specimens of epithelial ovarian cancer stages I–IV. Survival of patients was investigated using uni- and multivariate analysis.Results. Strong KAI1 expression was observed in 17.8% of cases, moderate in 27.1%, weak in 21.5%, and complete loss of KAI1 expression in 33.6%. Overexpression of p53 protein was observed in 45.8%. There was correlation of KAI1 expression neither with p53 expression nor with various clinical and histopathological parameters. Serous ovarian cancers showed significantly decreased staining intensity of KAI when compared to other histological types (P = 0.007). Univariate and multivariate analysis revealed that patients with strong or moderate expression of KAI1 had a significantly longer overall (P = 0.0013) and disease-free survival (P = 0.0048) when compared to those with low or absent expression.Conclusion. KAI1 downregulation is an independent prognostic factor in epithelial ovarian cancer, indicating dismal prognosis. Our study did not reveal a correlation between p53 status and KAI1 expression, suggesting that p53-independent mechanisms might be involved in the downregulation of KAI1.

Impact of human papillomavirus infection on the expression of the KAI1 metastasis suppressor protein in invasive cervical cancer

Downregulation of KAI1 metastasis suppressor protein is associated with dismal prognosis in a variety of cancers. Mutation of p53 was suggested to be involved in KAI1-downregulation. In cervical cancer, p53 is inactivated by human papillomavirus (HPV) oncoprotein E6 with the grade of inactivation depending on the HPV type. KAI1-expression was immunohistochemically determined in 67 specimens of cervical cancer, HPV-typing was performed using polymerase chain reaction (PCR), cloning, and sequencing. KAI1-downregulation was found in 68.1% of patients, HPV-infection in 91%. There was no association of KAI1-downregulation and infection with a particular HPV type. KAI1-downregulation in cervical cancer seems independent of HPV-E6 induced p53 inactivation.

Metastasis-Suppressor Genes: a Review and Perspective on an Emerging Field

Metastasis is the most lethal attribute of a cancer. There is a critical need for markers that will distinguish accurately those histologic lesions and disseminated cells with a high probability of causing clinically important metastatic disease from those that will remain indolent. While the development of new diagnostic markers of metastasis was the initial motivation for many studies, the biologic approach used to identify metastasis-suppressor genes has provided surprising insights into the in vivo mechanisms regulating the formation of metastases. This review and perspective describes the evolving view of the mechanisms that regulate metastasis and the importance of metastasis-suppressor genes in this process. The known metastasis-suppressor proteins or genes and the microcell-mediated chromosomal transfer strategy used to identify many of them are reviewed. New evidence for the role of these metastasis-suppressor proteins or genes in regulating the growth of disseminated cancer cells at the secondary site, the potential for the identification of novel therapeutic targets, and the multidisciplinary approach needed to translate this information into clinical tools for the treatment of metastatic disease are discussed.

Complementary DNA Encoding nm23/NDP Kinase Gene from the Korean Tiger Shark Scyliorhinus torazame.

: A new tumor suppressor gene, snm23, homologous to the gene for human nucleoside diphosphate kinase nm23/NDP was first cloned from Korean tiger shark (Scyliorhinus torazame) skin lambda ZAP-II complementary DNA library. The gene (named snm23) containing the tumor metastasis suppressor protein was sequenced. The nucleotide and deduced amino acid sequences of snm23 revealed an open reading frame of 450 bp that corresponded to a protein of 150 amino acid residues, with a calculated molecular mass of 16.8 kDa. Sequence comparison of snm23 with nm23/NDP kinases was performed. In order to determine tissue specificity, reverse transcription-polymerase chain reaction was used. The expression of snm23/NDP kinase was detected in tissues from skin, cartilage, and liver of Korean tiger shark.

Glyceraldehyde-3-phosphate Dehydrogenase and Nm23-H1/Nucleoside Diphosphate Kinase A: TWO OLD ENZYMES COMBINE FOR THE NOVEL Nm23 PROTEIN PHOSPHOTRANSFERASE FUNCTION

We have recently discovered an alternative function of the putative metastasis suppressor protein Nm23, which is identical to nucleoside diphosphate kinase, as a protein phosphotransferase in vitro. While purified native Nm23 protein did not phosphorylate other proteins, we could purify a Nm23-associated protein that activates the protein phosphotransferase function; it was identified as a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) isoenzyme. Co-expression and purification of (His)6-tagged GAPDH in combination with either Nm23-H1 or Nm23-H2 in baculovirus-infected Sf9 cells showed that only Nm23-H1, but not Nm23-H2, forms a stable complex with GAPDH. Protein phosphotransferase activity was confirmed for the recombinant GAPDH.Nm23-H1 complex but not for either of the enzymes alone, nor was this activity observed after simple mixing of the purified proteins in vitro. The molecular mass of the highly purified recombinant GAPDH.Nm23-H1 complex suggests that a dimer of GAPDH interacts with a dimer of Nm23-H1. In contrast to the complex with GAPDH, co-expression of Nm23-H1 with antioxidant protein (MER-5) or creatine kinase did not activate the protein phosphotransferase function, indicating that this activation may specifically require GAPDH as a binding partner.

Reduction of translation initiation factor 4E decreases the malignancy of ras-transformed cloned rat embryo fibroblasts.

Expression of the T24ras oncogene induces malignancy (tumor growth, invasion and metastasis) in cloned rat embryo fibroblasts (CREF T24). In CREF T24, the rate of phosphorylation of eukaryotic translation initiation factor 4E (eIF-4E) is increased, resulting in increased protein synthesis rates. We have recently shown that reducing the protein levels of eIF-4E in CREF T24 (AS4E line) markedly decreases soft-agar colonization, increases tumor latency periods and increases tumor doubling times without significantly altering monolayer growth. In this study, cells with reduced eIF-4E had delayed and reduced invasiveness and decreased experimental metastasis. Furthermore, reduced eIF-4E levels correlated with decreased expression of the metastasis-associated 92-kDa collagenase type-IV and exon-6 variants of the CD44 adhesion molecule [CD44(6v)]. Reduced eIF-4E levels correlated inversely with increased levels of the putative metastasis-suppressor protein nm23. Cell lines established from AS4E tumors and lung metastases exhibited increased levels of eIF-4E protein and protein synthesis rates compared to the AS4E line. Tumor-derived AS4E had the shortened tumor latency periods of CREF T24 but displayed the slow tumor-growth rates of AS4E. Tumor-derived AS4E exhibited the metastatic capacity of CREF T24 controls. Furthermore, tumor- and lung-nodule-derived AS4E expressed levels of CD44 (6v) and the 92-kDa collagenase type IV comparable to CREF T24 and displayed reduced levels of nm23 relative to AS4E. These results demonstrate that eIF-4E is an important effector molecule involved in oncogenic p21ras-induced malignant transformation.

Differential nm23 gene expression at the fetal-maternal interface.

The product of the nm23 gene has been proposed as a candidate tumour metastasis suppressor protein. A strong association has been observed between reduced expression of the nm23 gene and acquisition of metastatic behaviour in some tumour cells, including breast cancer and melanoma, but not in others, such as neuroblastoma and colon, cervical and thyroid cancers. During the early gestation period both human and murine trophoblast cells exhibit in vitro invasive properties similar to those of neoplastic cells. Such invasive properties, however, disappear in the late stage of gestation. In the present study, we examined the abundance of nm23 mRNA from various fetal-maternal interface tissues (uterus, decidua, placenta and embryo) during early (day 8), mid (day 14) and late (day 18) stages of gestation in CD1 mice, in order to determine whether nm23 plays any anti-invasive and/or biological roles during gestation. nm23 was found to be expressed in all the tissues during the early and mid stages of gestation. The expression levels were, however, variable among different tissues and development stages. In the early stage, nm23 mRNA levels were the highest and similar among tissues from the uterus, decidua, placenta and embryo. In the mid stage, the mRNA levels were reduced significantly in the uterus, decidua and placenta, but not in the embryo. In the late stage, nm23 mRNA was further reduced to the extent that it could not be seen in the decidua, was barely seen in the uterus and was weakly present in the placenta. However, the mRNA level of the embryo in the late stage was still high and similar to the early stage. We also examined nm23 expression in trophoblast cells from normal human term placenta and a highly metastatic human choriocarcinoma cell line, JAR. nm23 expression was significantly higher in JAR than in normal placenta, indicating that nm23 does not appear to have an anti-metastatic function in this cell line. Several cytokines--interleukin 2 (IL-2), tumour necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma)--and prostaglandin E2 (PGE2) known to modulate tumour growth and metastasis were examined to determine whether they regulate nm23 expression in JAR in vitro. The B16F10 melanoma cell line was used as control. No effect was found in the JAR cell line, whereas TNF-alpha, IFN-gamma and PGE2 down-regulated nm23 expression in the B16F10 cell line.(ABSTRACT TRUNCATED AT 400 WORDS)

A serine phosphorylation of Nm23, and not its nucleoside diphosphate kinase activity, correlates with suppression of tumor metastatic potential.

We describe a serine phosphorylation of the putative metastasis suppressor protein Nm23, and present evidence of its relevance to the signal transduction and tumor metastatic processes. Nm23 was previously demonstrated to exhibit nucleoside diphosphate kinase (NDPK) activity, which transfers a phosphate among nucleoside tri- and diphosphates via an Nm23-phospho-histidine intermediate. Recent data have dissociated the NDPK activity of Nm23 from its phenotypic effects; therefore we have asked whether Nm23 possesses additional biochemical functions. An acid-stable (nonhistidine) phosphorylation was identified on autophosphorylated purified recombinant Nm23 proteins and [32P]orthophosphate-labeled human breast carcinoma and murine melanoma Nm23. Phosphoamino acid analysis identified serine as the acid-stable phosphorylation and serine 44 as the major site of phosphorylation. The acid stable phosphorylation (serine) of Nm23 was inhibited by cAMP in vitro and forskolin in vivo, suggesting that this phosphorylation pathway is regulated in signal transduction. No effect of cAMP was observed on Nm23 NDPK activity. Once phosphorylated, Nm23-phosphoserine can release free phosphate in vitro. The biological relevance of the novel phosphorylation identified herein is suggested by the direct correlation of in vivo Nm23 acid-stable phosphorylation levels, but not Nm23 NDPK activity, with suppression of tumor metastatic potential among control and nm23-1 transfected murine melanoma cells.

High levels of Nm23 gene expression in advanced stage of thyroid carcinomas

The product of Nm23 gene has been proposed as a candidate tumour metastasis suppressor protein. A strong association has been observed between reduced expression of Nm23 gene and acquisition of metastatic behaviour in some tumour cells including breast cancer and melanoma, but not in others such as colon cancer, neuroblastoma, and cervical cancer. In the present study, we examined the abundance of Nm23 mRNA in 39 thyroid tissue specimens including five multinodular goitres, one follicular adenoma, 26 papillary and three follicular carcinomas, and four anaplastic carcinomas. Nm23 was found to be expressed in all the tissue specimens. The expression was, however, variable in different stages of thyroid carcinoma. In stages I through III of differentiated thyroid carcinoma, the average level of Nm23 gene expression was comparable to that in multinodular goitres. In advanced stage of thyroid carcinoma (stage IV and anaplastic), 2-fold increase of Nm23 expression was noted. No mutations were found in the coding region of the gene. Nm23 mRNA level cannot, therefore, be used as a marker of low metastatic potential in thyroid carcinomas. The association of high level Nm23 expression with anaplastic thyroid carcinoma suggests its correlation with rapid cell proliferation.