Metastasis-suppressing Activity Research Articles

Abstract 3106: Elucidating molecular mechanisms linking microRNA-206 loss to tumor progression and metastasis

Abstract Metastasis is the leading cause of cancer-related deaths, and this occurs when tumor cells invade basement membranes and blood vessels to colonize other tissues. Metastasis is facilitated by extracellular matrix (ECM)-degrading structures called invadopodia. Recently, several microRNAs (miRNAs) have been identified as tumor and metastasis suppressor genes. Loss of expression of miR-206 has been correlated with poor prognosis in gastric cancer, breast cancer, rhabdomyosarcoma and melanoma. A previous study by others has demonstrated that restoring miR-206 expression in human breast cancer cells was sufficient to block metastasis in tumor xenograft assays. However, the targets of miR-206 that cause metastasis in these cancer models have not been fully characterized. Here, we have tested the effects of miR-206 on invadopodia in metastatic cancer cells, and identified a novel target that may explain this effect. Transducer of Cdc42-mediated Actin assembly-1 (Toca-1) is an adaptor protein that promotes formation of invadopodia and metastasis in breast cancer models, and has a predicted miR-206 binding site within the 3′ UTR of Toca-1 transcripts. To test whether the high levels of Toca-1 expression that we have observed in metastatic cancer cell lines was due to loss of miR-206, we rescued miR-206 expression using transient, stable or inducible approaches in MDA-MB-231 breast cancer, A375 melanoma, and H1299 lung cancer cell lines. In each cell model, Toca-1 expression was reduced upon miR-206 expression at both the mRNA and protein levels, thus validating Toca-1 as a new target of miR-206. Further testing of the phenotypes induced by miR-206 rescue revealed dramatic defects in ECM degradation and cell invasion in breast cancer and melanoma cell models. In subcutaneous tumor xenograft assays, stable rescue of miR-206 in H1299 tumors led to defects in tumor growth compared to those expressing a scrambled control miRNA. In addition, miR-206 expression caused a dramatic reduction in the numbers of lung metastases. We are currently extending this to our melanoma models and inducible models to define changes in metastasis-related targets of miR-206 that are aberrantly expressed in metastatic cancers. In conclusion, our study demonstrates for the first time a link between miR-206 and suppression of invadopodia formation via Toca-1 silencing, which likely contributes to the metastasis suppressing activity of miR-206 in multiple cancer types. Citation Format: Kathleen D. Watt, Peter Truesdell, Andrew W. Craig. Elucidating molecular mechanisms linking microRNA-206 loss to tumor progression and metastasis. [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 3106. doi:10.1158/1538-7445.AM2015-3106

Abstract 517: Ectopic expression of an obscurin signaling cassette decreases migration and invasion of metastatic breast cancer cells.

Abstract The obscurins (∼70-820 kDa) are a family of proteins expressed from the 150 kb OBSCN gene, located on human chromosome 1. Sequencing analysis of breast and colorectal cancers, as well as melanoma and glioblastoma, revealed that obscurins are mutated at a substantial frequency and these mutations may be driving tumor formation. Our work has recently demonstrated that obscurins are present in normal breast, skin, and colon cells, but absent in their cancerous counterparts. Downregulation of obscurins’ expression is sufficient to allow non-tumorigenic breast epithelial cells to evade apoptosis induced by etoposide. Furthermore, absence of obscurins allows increased migration and invasion of breast epithelial cells. Remarkably though, we observed that ectopic expression of a fragment of obscurin composed of tandem src homology 3-Rho guanine exchange factor-pleckstrin homology (SH3-RhoGEF-PH) motifs was sufficient to decrease monolayer migration and invasion of metastatic breast cancer MDA-MB-231 cells, which are naturally deficient in obscurins, by >20% and >70%, respectively. Based on our observations that 1) absence of obscurins allows increased migration and invasion of breast cells, and 2) expression of an obscurin cassette mitigates this migratory and invasive behavior of metastatic breast cancer cells, we postulate that obscurins may act as metastasis suppressors in breast epithelium. We have therefore begun to investigate how the tripartite obscurin fragment, SH3-RhoGEF-PH, can influence the behavior of the cytoskeleton and therefore affect migration and invasion. We have found that the RhoGEF domain directly induces guanine nucleotide exchange in RhoA, but not Rac1 or Cdc42. This activity may be allosterically regulated by the adjacent SH3 domain, which interacts with the RhoGEF domain in a yeast two-hybrid assay. Furthermore, the PH domain binds to 3’-phosphorylated inositol lipid headgroups, providing a mechanism for the obscurins to respond to signals generated at the plasma membrane. Together, this evidence suggests that the tripartite signaling cassette can recapitulate the full-length obscurins’ metastasis-suppressing activity, and may merit development as a therapy for advanced breast cancer. Citation Format: Nicole A. Perry, Aikaterini Kontrogianni-Konstantopoulos. Ectopic expression of an obscurin signaling cassette decreases migration and invasion of metastatic breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 517. doi:10.1158/1538-7445.AM2013-517

Abstract 553: Interactions among JNK, p38, Akt and canonical Wnt pathways in murine mammary adenocarcinoma cells reexpressing Glypican-3 (GPC3).

Abstract GPC3 is expressed in normal mammary tissues, but it disappears in tumors. We have transfected the GPC3 gene into the murine mammary adenocarcinoma LM3 cells, demonstrating that it acts as a metastasis suppressor. We have also reported that LM3-GPC3 cells exhibit an inhibition of Akt and canonical Wnt pathways, while they show an activation of non-canonical Wnt (JNK) and p38 signaling. The aim of this work was to study the interactions among GPC3 modulated pathways. For this, subconfluent LM3-GPC3 cells were treated with SB203589 or SP600125 (p38 and JNK pharmacological inhibitors, respectively), or with the vehicle. Also, we introduced by viral infection a constitutively active variant of Akt1 (CA Akt), or the empty vector, into LM3-GPC3 cells. The effect of the different treatments on the activity of the other pathways was evaluated by WB. We demonstrated that in addition to the central role of JNK in the non-canonical Wnt pathway, it also regulates canonical signaling. We found that SP600125 induced a 2 to 5-fold increase in the cytoplasmic β-Catenin levels, as well it reduced 2 to 6 times E-Cadherin expression. Therefore, our results suggest that JNK activation would be required for the canonical Wnt signaling inhibition induced by GPC3. On the other side, p38 pathway is considered independent of Wnt signaling. In association, SB203589 did not modulate either cytoplasmic β-Catenin or E-Cadherin levels, suggesting that p38 activation would not participate in the canonical Wnt pathway inhibition induced by GPC3. We also observed that LM3-GPC3 cells expressing CA Akt, showed less E-Cadherin than controls. So, Akt inhibition would be essential to increase of E-Cadherin expression induced by GPC3. In relation with the non-canonical Wnt pathway, we found that SB203589 induced a partial decrease in phospho-JNK levels. Therefore, p38 would be involved in the non-canonical Wnt signaling activation induced by GPC3. Inhibitors actions on Akt pathway were analyzed. We determined that SP600125 had no effect on Akt activation, suggesting that Akt inhibition induced by GPC3 is independent of non-canonical Wnt signaling. On the other hand, although without reaching LM3-vector levels, SB203589 caused a 2-fold increase in phospho-Akt levels. This indicates that the activation of p38 would be necessary to induce the inhibition of Akt signaling detected in GPC3 reexpressing cells. In sum, we have dissected some of the functional and hierarchical interactions between the pathways modulated by GPC3 that may contribute to the metastasis suppressing activity of this proteoglycan. Citation Format: Maria Giselle Peters, Rocio Tascon, Lilian F. Castillo, Elisa Bal de Kier Joffe. Interactions among JNK, p38, Akt and canonical Wnt pathways in murine mammary adenocarcinoma cells reexpressing Glypican-3 (GPC3). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 553. doi:10.1158/1538-7445.AM2013-553

Anti-S100A4 Antibody Suppresses Metastasis Formation by Blocking Stroma Cell Invasion

The small Ca-binding protein, S100A4, has a well-established metastasis-promoting activity. Moreover, its expression is tightly correlated with poor prognosis in patients with numerous types of cancer. Mechanistically, the extracellular S100A4 drives metastasis by affecting the tumor microenvironment, making it an attractive target for anti-cancer therapy. In this study, we produced a function-blocking anti-S100A4 monoclonal antibody with metastasis-suppressing activity. Antibody treatment significantly reduced metastatic burden in the lungs of experimental animals by blocking the recruitment of T cells to the site of the primary tumor. In vitro studies demonstrated that this antibody efficiently reduced the invasion of T cells in a fibroblast monolayer. Moreover, it was capable of suppressing the invasive growth of human and mouse fibroblasts. We presume therefore that the antibody exerts its activity by suppressing stroma cell recruitment to the site of the growing tumor. Our epitope mapping studies suggested that the antibody recognition site overlaps with the target binding interface of human S100A4. We conclude here that this antibody could serve as a solid basis for development of an efficient anti-metastatic therapy.

Abstract A6: microRNA sequencing of AKXD recombinant inbred panel identifies miR-216b as a candidate metastasis suppressor in a murine model of breast cancer

Abstract Metastatic disease continues to be the primary cause of mortality in cancer. Using a combination of complex genetics, mouse models of breast cancer, and human epidemiology, our laboratory has demonstrated that germline polymorphism contributes to an individual's susceptibility to metastasis. In the current study, we have used the AKXD recombinant inbred panel, which is derived from inbred mice of metastasis resistant, DBA, and metastasis-prone, AKR, genetic backgrounds, to identify microRNAs whose expression is associated with metastasis. microRNA sequencing of tumor tissue from the progeny of AKXD x Polyoma Middle-T mice demonstrated that the expression of the miR-216/217 cluster is significantly negatively correlated with pulmonary metastases in the AKXD panel. Linkage analysis demonstrated a significant association between the miR-216/217 locus and the DBA allele, suggesting that the difference in expression is inherited rather than somatically acquired. Finally, we show that ectopic expression of miR-216b in murine mammary tumor cells suppresses pulmonary metastasis without significantly impacting primary tumor growth in vivo. These findings suggest that miR-216b is an inherited modifier of metastasis with metastasis suppressing activity in a mouse model of breast cancer. Citation Format: Farhoud Faraji, Ying Hu, Natalie Goldberger, Gang Wu, Ken H. Buetow, Jinghui Zhang, Kent W. Hunter. microRNA sequencing of AKXD recombinant inbred panel identifies miR-216b as a candidate metastasis suppressor in a murine model of breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr A6.

Abstract C11: Relationship between GLIPR-1 and c-Myc expression in progression of gastric cancer.

Abstract Glioma Pathogenesis Related-Protein (GLIPR)-1 is up-regulated by p53 and has proapoptotic, antiangiogenic, immunostimulatory and metastasis-suppressing activity in prostate cancer. How ever its expression and biological role is not well known in gastric cancer. Meanwhile, c-Myc overexpression has shown to be related to on the cell growth, proliferation, invasion and cell cycle regulation in gastric cancer. Here, we investigated the expression pattern of GLIPR-1 and its relation with c-Myc expression in gastric cancer. The protein expressions of GLIPR-1 and c-Myc were evaluated by western blot with whole lysate of 39 gastric cancer cell lines. Band intensities were measured by Image J program. By Immunohistochemisty, we checked the expression of GLIPR-1 in formalin-fixed paraffin-embedded gastric cancer tissues of stage Ito IVwith 20 samples for each stage. GLIPR-1 was overexpressed in 20 of 39 (51.3%) and c-Myc was overexpressed in 23 of 39 (60%) gastric cancer cell lines. 16 of 39 cell lines (41.1%) had co-expression. Level of GLIPR-1 expression was higher in cell lines established from primary site of gastric cancer (n=13) than the cell lines from metastasis site (n=26) (p < 0.01). On the other hand, c-Myc was overexpressed in the cell lines of metastasis site than primary sites (p<0.01). GLIPR-1 expression of two group cell lines was negatively correlated with c-Myc expression (R = −0.48). In IHC, GLIPR-1 was expressed in nucleus and cytosol of gastric cancer cells, and GLIPR-1 expression was stage-specifically reduced. This study showed that GLIPR-1 is downregulated while c-Myc is overexpressed in advanced gastric cancer, ssuggestig their associative role in gastric cancer. In addition, as GLIPR-1 is decreased throughout the disease progression. GLIPR-1 might be the specific therapeutic target for advanced gastric cancer. Also, there is the potential to develop GLIPR-1 and c-Myc expression as prognostic biomarker for gastric cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C11.

Control of Protein Kinase C Activity, Phorbol Ester-induced Cytoskeletal Remodeling, and Cell Survival Signals by the Scaffolding Protein SSeCKS/GRAVIN/AKAP12

The product of the SSeCKS/GRAVIN/AKAP12 gene ("SSeCKS") is a major protein kinase (PK) C substrate that exhibits tumor- and metastasis-suppressing activity likely through its ability to scaffold multiple signaling mediators such as PKC, PKA, cyclins, calmodulin, and Src. Although SSeCKS and PKCα bind phosphatidylserine, we demonstrate that phosphatidylserine-independent binding of PKC by SSeCKS is facilitated by two homologous SSeCKS motifs, EG(I/V)(T/S)XWXSFK(K/R)(M/L)VTP(K/R)K(K/R)X(K/R)XXXEXXXE(E/D) (amino acids 592-620 and 741-769). SSeCKS binding to PKCα decreased kinase activity and was dependent on the two PKC-binding motifs. SSeCKS scaffolding of PKC was increased in confluent cell cultures, correlating with significantly increased SSeCKS protein levels and decreased PKCα activity, suggesting a role for SSeCKS in suppressing PKC activation during contact inhibition. SSeCKS-null mouse embryo fibroblasts displayed increased relative basal and phorbol ester (phorbol 12-myristate 13-acetate)-induced PKC activity but were defective in phorbol 12-myristate 13-acetate-induced actin cytoskeletal reorganization and cell shape change; these responses could be rescued by the forced expression of full-length SSeCKS but not by an SSeCKS variant deleted of its PKC-binding domains. Finally, the PKC binding sites in SSeCKS were required to restore cell rounding and/or decreased apoptosis in phorbol ester-treated LNCaP, LNCaP-C4-2, and MAT-LyLu prostate cancer cells. Thus, PKC-mediated remodeling of the actin cytoskeleton is likely regulated by the ability of SSeCKS to control PKC signaling and activity through a direct scaffolding function.

MDM2 interacts with NME2 (non-metastatic cells 2, protein) and suppresses the ability of NME2 to negatively regulate cell motility

MDM2 expression, combined with increased p53 expression, is associated with reduced survival in several cancers, but is particularly of interest in renal cell carcinoma (RCC) where evidence suggests the presence of tissue-specific p53/MDM2 pathway defects. We set out to identify MDM2-interacting proteins in renal cells that could act as mediators/targets of MDM2 oncogenic effects in renal cancers. We identified the non-metastatic cells 2, protein; NME2 (NDPK-B, NM23-B/-H2), a nucleoside diphosphate kinase, as an MDM2-interacting protein using both a proteomic-based strategy [affinity chromatography and tandem mass spectrometry [MS/MS] from HEK293 cells] and a yeast two-hybrid screen of a renal carcinoma cell-derived complementary DNA library. The MDM2-NME2 interaction is highly specific, as NME1 (87.5% amino acid identity) does not interact with MDM2 in yeast. Specific NME proteins display well-documented cell motility and metastasis-suppressing activity. We show that NME2 contributes to motility suppression under conditions where MDM2 is expressed at normal physiological/low levels. However, up-regulation of MDM2 in RCC cells abolishes the ability of NME2 to suppress motility. Significantly, when MDM2 expression is down-regulated in these cells using small interfering RNA, the motility-suppressing activity of NME2 is rescued, confirming that MDM2 expression causes the loss of NME2 cell motility regulatory function. Thus MDM2 up-regulation in renal cancer cells can act in a dominant manner to abrogate the function of a potent suppressor of motility and metastasis. Our studies identify a novel protein-protein interaction between MDM2 and NME2, which suggests a mechanism that could explain the link between MDM2 expression and poor patient survival in RCC.

Role for transcription factor TFII‐I in the suppression of SSeCKS/Gravin/Akap12 transcription by Src

The SSeCKS/Gravin/AKAP12 gene, encoding a kinase scaffolding protein with metastasis-suppressing activity, is transcriptionally downregulated in Src-transformed cells through the recruitment of HDAC1 to a Src-responsive proximal promoter site charged with Sp1, Sp3 and USF1. However, the ectopic expression of these proteins formed a suppressive complex in Src-transformed but not in parental NIH3T3 cells, suggesting the involvement of additional repressor factors. Transcription factor II-I (TFII-I) [general transcription factor 2i (Gtf2i)] was identified by mass spectrometry as being associated with the SSeCKS promoter complex in NIH3T3/Src cells, and moreover, the Src-induced tyrosine phosphorylation of TFII-I significantly increased its binding to the SSeCKS proximal promoter. siRNA-mediated knockdown of TFII-I or the expression of TFII-I(Y248/249F) caused the derepression of SSeCKS in NIH3T3/Src cells. Taken with previous data showing that the tyrosine phosphorylation of TFII-I facilitates its nuclear translocation, these data suggest that Src-family kinase-mediated phosphorylation converts a portion of TFII-I into a transcriptional repressor.

Rb-dependent cellular senescence, multinucleation and susceptibility to oncogenic transformation through PKC scaffolding by SSeCKS/AKAP12

A subset of AKAPs (A Kinase Anchoring Proteins) regulate signaling and cytoskeletal pathways through the spaciotemporal scaffolding of multiple protein kinases (PK) such as PKC and PKA, and associations with the plasma membrane and the actin-based cytoskeleton. SSeCKS/Gravin/Akap12 expression is severely downregulated in many advanced cancers and exhibits tumor- and metastasis-suppressing activity. akap12-null (KO) mice develop prostatic hyperplasia with focal dysplasia, but the precise mechanism how Akap12 prevents oncogenic progression remains unclear. Here, we show that KO mouse embryonic fibroblasts (MEF) exhibit premature senescence marked by polyploidy and multinucleation, and by increased susceptibility to oncogenic transformation. Although p53 and Rb pathways are activated in the absence of Akap12, senescence is dependent on Rb. Senescence is driven by the activation of PKCα, which induces p16Ink4a/Rb through a MEK-dependent downregulation of Id1, and PKCδ, which downregulates Lats1/Warts, a mitotic exit network kinase required for cytokinesis. Our data strongly suggest that Akap12 controls Rb-mediated cell aging and oncogenic progression by directly scaffolding and attenuating PKCα/δ.

G-helix of Maspin Mediates Effects on Cell Migration and Adhesion

Maspin is a member of the serine protease inhibitor (serpin) superfamily that lacks protease inhibitory ability, although displaying tumor metastasis-suppressing activity resulting from its influence on cell migration, invasion, proliferation, apoptosis, and adhesion. The molecular mechanisms of these actions of maspin are as yet undefined. Here, we sought to identify critical functional motifs by the expression of maspin with point mutations at sites potentially involved in protein-protein interactions: the G α-helix (G-helix), an internal salt bridge or the P1 position of the reactive center loop. Our findings indicate that only mutations in the G-helix attenuated inhibition of cell migration by maspin and that this structural element is also involved in the effect of maspin on cell adhesion. The action of maspin on cell migration could be mimicked by a 15-mer G-helix peptide, indicating that the G-helix is both essential and sufficient for this effect. In addition, we provide evidence that the effects of the G-helix of maspin are dependent on β1 integrins. These data reveal that the major extracellular functions associated with the tumor suppressive action of maspin likely involve interactions in which the G-helix plays a key role.

GLIPR1/RTVP-1 tumor suppressor gene expressed by adenovirus vector as neoadjuvant intraprostatic injection for localized high-risk prostate cancer preceding prostatectomy.

e15026 Background: GLIPR1/RTVP-1 is up-regulated by p53 in prostate cancer (PCa) cells and has preclinical pro-apoptotic, anti- angiogenic, immunostimulatory and metastasis-suppressing activity. A ...

Inverse Correlation of NM23 Expression with Lysophosphatidic Acid Receptor EDG2/lpa1 Expression of Human Leukemia Cells during Myeloid Differentiation Induced by All-Trans Retinoic Acid

Overexpression of the nm23 gene is found in many hematological malignancies, and predicts poor treatment outcome. Because the amount of intracellular NM23-H1 protein is inversely correlated with differentiation and exogenous over expression of the nm23-H1 in myeloid leukemia cells reduced the sensitivity to induction of myeloid differentiation, nm23-overexpression is considered to function as a differentiation-suppressor. However, the molecular mechanism of this phenomenon is unknown. Recently, lysophosphatidic acid (LPA) receptor EDG2/lpa1 was identified as a gene down-regulated by nm23-H1 and associated with cell motility/metastasis suppressor functions in breast cancer cells. Although the clinical significance of nm23-H1 overexpression in leukemia is completely opposite to that in breast cancer, there might be a common molecular mechanism between the metastasis-suppressing activity and differentiation-inhibiting activity of nm23-H1 overexpression, because many functional characteristics of macrophages/neutrophils are similar to those of motile and metastatic tumor cells. Here, we examined the EDG2/lpa1 expression level and its association with NM23 expression level and myeloid differentiation of leukemia cells. EDG2 and NM23 proteins were expressed at different levels in all leukemia cells tested (HL-60, NB4, THP-1, U937, K562, HEL, BALM-1, BALM-3, MOLT4, Jurkat). During all-trans retinoic acid (ATRA)-induced myeloid differentiation of HL-60 cells, the NM23 expression decreased and EDG2 expression inversely increased. Similar effects of ATRA were observed in myeloid differentiation of NB4 and THP-1 cells. NM23 expression levels and EDG2 expression levels modulated by ATRA treatment were inversely correlated (Spearman's correlation coefficient, rs = −0.7538, p = 0.0237). However, these correlations were not found in the erythroid differentiation of ATRA-treated HEL and K562 cells. Cell motility is required for myeloid differentiation; therefore, there might be a common mechanism, the inhibition of cell motility thorough EDG2 down-regulation by NM23-H1 overexpression, between differentiation-inhibiting activity in leukemia cells and motility-inhibiting activity in breast cancer cells. The discrepancy in the clinical significance of NM23-H1 overexpression between leukemia and breast cancer might be resolved.

Point mutations affecting the oligomeric structure of Nm23-H1 abrogates its inhibitory activity on colonization and invasion of prostate cancer cells

In order to identify Nm23-H1’s structural motifs influencing its metastasis-inhibitory activity, we transfected DU 145 human prostate carcinoma cells with the expression vector encoding the Nm23-H1 protein with mutations at the following amino acids: serine-44, a phosphorylation site; proline-96, a site corresponding to the k-pn mutation that causes developmental defects in Drosophila; and serine-120, a site of mutation in human neuroblastoma and phosphorylation. Significant decrease in colonization in soft agar and invasiveness of DU 145 cells was observed in the wild type nm23-H1 transfectants, and also in the serine-44 and serine-120 to alanine mutant nm23-H1-transfected cell lines. However, the k-pn type proline-96 to serine (P96S) and neuroblastoma type serine-120 to glycine (S120G) mutations of Nm23-H1 abrogated its inhibitory activity on colonization and invasion. Meanwhile, all of the recombinant mutant Nm23-H1 proteins produced in Escherichia coli exhibited NDP kinase activity levels at the wild type protein, although the P96S and S120G mutant proteins exhibited decreased histidine protein kinase activity and autophosphorylation level, respectively. Interestingly, only two of the mutant recombinant Nm23-H1 proteins examined, P96S and S120G, exhibited reduced hexameric and increased dimeric oligomerization relative to the wild type. These correlative data suggest that the metastasis-suppressing activity of Nm23-H1 may depend on its oligomeric structure, but not on its NDP kinase activity.

Differential expression of the nm23 protein in the progression of oesophageal adenocarcinoma

Aim Some studies have shown that abnormalities of the nm23 gene or its expression may be important in tumour dissemination, suggesting that the gene may have metastasis suppressing activity. This study set out to determine if nm23 protein expression is altered with progression and dissemination in oesophageal adenocarcinoma. Methods Paraffin-embedded, archival tissues of surgical resection specimens of oesophageal adenocarcinoma (n = 46), some of which were accompanied by tissue from areas with high-grade dysplasia (n = 24) and from metastasis in regional lymph nodes (n = 16) were studied. Histologically normal oesophageal glandular tissue (of cardiac-type) (n = 32) obtained from areas of the resections located away from the primary tumour masses and archival tissues of Barrett's metaplasia obtained from endoscopic biopsies (n = 77) were used as non-neoplastic controls. Sections were immuno-histochemically stained by the labelled streptavidin-biotin method using NCL-nm23 antibody. Results The total or overall amount of nm23 protein expression paralleled that of cytoplasmic expression and was increased in oesophageal adenocarcinomas (36/46 cases, 78%) when compared with normal oesophageal glandular epithelium (2/32, 6%), Barrett's metaplasia (8/77, 10%) and dysplasia (14/24, 58%). In metastatic carcinoma in regional lymph nodes, overall nm23 expression was similar in proportion (13/16, 81%) to that seen in primary carcinoma. In the analysis of the sequential development of oesophageal adenocarcinoma based on non-neoplastic, preneoplastic and neoplastic archival material, it was found that a high level of overall nm23 expression occurred firstly at the transition from Barrett's metaplasia (8/77, 10%) to dysplasia (14/24, 58%). Nuclear nm23 expression was low in dysplastic tissue (with none of the cases having a high level of nuclear nm23 expression) followed by increased levels as the lesion progressed to invasive adenocarcinoma (13/46, 28%) and metastatic carcinoma in regional lymph nodes (10/16, 63%). However, nm23 expression did not appear to correlate with sex, age, tumour size, extent of tumour infiltration into the oesophageal wall, presence of lymph node metastasis or overall patient survival. Conclusion An overall increase in nm23 expression or increase in nm23 expression in the cytoplasm of cells may be important in the early development of oesophageal adenocarcinoma but increased levels of nuclear nm23 occur in its progression to metastatic disease. Some studies have shown that abnormalities of the nm23 gene or its expression may be important in tumour dissemination, suggesting that the gene may have metastasis suppressing activity. This study set out to determine if nm23 protein expression is altered with progression and dissemination in oesophageal adenocarcinoma. Paraffin-embedded, archival tissues of surgical resection specimens of oesophageal adenocarcinoma (n = 46), some of which were accompanied by tissue from areas with high-grade dysplasia (n = 24) and from metastasis in regional lymph nodes (n = 16) were studied. Histologically normal oesophageal glandular tissue (of cardiac-type) (n = 32) obtained from areas of the resections located away from the primary tumour masses and archival tissues of Barrett's metaplasia obtained from endoscopic biopsies (n = 77) were used as non-neoplastic controls. Sections were immuno-histochemically stained by the labelled streptavidin-biotin method using NCL-nm23 antibody. The total or overall amount of nm23 protein expression paralleled that of cytoplasmic expression and was increased in oesophageal adenocarcinomas (36/46 cases, 78%) when compared with normal oesophageal glandular epithelium (2/32, 6%), Barrett's metaplasia (8/77, 10%) and dysplasia (14/24, 58%). In metastatic carcinoma in regional lymph nodes, overall nm23 expression was similar in proportion (13/16, 81%) to that seen in primary carcinoma. In the analysis of the sequential development of oesophageal adenocarcinoma based on non-neoplastic, preneoplastic and neoplastic archival material, it was found that a high level of overall nm23 expression occurred firstly at the transition from Barrett's metaplasia (8/77, 10%) to dysplasia (14/24, 58%). Nuclear nm23 expression was low in dysplastic tissue (with none of the cases having a high level of nuclear nm23 expression) followed by increased levels as the lesion progressed to invasive adenocarcinoma (13/46, 28%) and metastatic carcinoma in regional lymph nodes (10/16, 63%). However, nm23 expression did not appear to correlate with sex, age, tumour size, extent of tumour infiltration into the oesophageal wall, presence of lymph node metastasis or overall patient survival. An overall increase in nm23 expression or increase in nm23 expression in the cytoplasm of cells may be important in the early development of oesophageal adenocarcinoma but increased levels of nuclear nm23 occur in its progression to metastatic disease.

Differential expression of the nm23 protein in the progression of oesophageal adenocarcinoma

Some studies have shown that abnormalities of the nm23 gene or its expression may be important in tumour dissemination, suggesting that the gene may have metastasis suppressing activity. This study set out to determine if nm23 protein expression is altered with progression and dissemination in oesophageal adenocarcinoma. Paraffin-embedded, archival tissues of surgical resection specimens of oesophageal adenocarcinoma (n=46), some of which were accompanied by tissue from areas with high-grade dysplasia (n=24) and from metastasis in regional lymph nodes (n=16) were studied. Histologically normal oesophageal glandular tissue (of cardiac-type) (n=32) obtained from areas of the resections located away from the primary tumour masses and archival tissues of Barrett's metaplasia obtained from endoscopic biopsies (n=77) were used as non-neoplastic controls. Sections were immunohistochemically stained by the labelled streptavidin-biotin method using NCL-nm23 antibody. The total or overall amount of nm23 protein expression paralleled that of cytoplasmic expression and was increased in oesophageal adenocarcinomas (36/46 cases, 78%) when compared with normal oesophageal glandular epithelium (2/32, 6%), Barrett's metaplasia (8/77, 10%) and dysplasia (14/24, 58%). In metastatic carcinoma in regional lymph nodes, overall nm23 expression was similar in proportion (13/16, 81%) to that seen in primary carcinoma. In the analysis of the sequential development of oesophageal adenocarcinoma based on non-neoplastic, preneoplastic and neoplastic archival material, it was found that a high level of overall nm23 expression occurred firstly at the transition from Barrett's metaplasia (8/77, 10%) to dysplasia (14/24, 58%). Nuclear nm23 expression was low in dysplastic tissue (with none of the cases having a high level of nuclear nm23 expression) followed by increased levels as the lesion progressed to invasive adenocarcinoma (13/46, 28%) and metastatic carcinoma in regional lymph nodes (10/16, 63%). However, nm23 expression did not appear to correlate with sex, age, tumour size, extent of tumour infiltration into the oesophageal wall, presence of lymph node metastasis or overall patient survival. An overall increase in nm23 expression or increase in nm23 expression in the cytoplasm of cells may be important in the early development of oesophageal adenocarcinoma but increased levels of nuclear nm23 occur in its progression to metastatic disease.

Nm23 protein expression in colorectal carcinoma metastasis in regional lymph nodes and the liver

Aims The nm23 gene has been shown to have metastasis suppressing activity and abnormalities of the gene or its expression may be important in tumour progression and dissemination. This study was set out to investigate the possible role of the nm23 in colorectal adenocarcinoma dissemination by examining the level of nm23 protein expression in colorectal carcinoma metastasis in regional lymph nodes and the liver. Methods Using a monoclonal antibody, NCL-nm23 (Novocastra), immunohistochemical expression of the nm23 protein was examined in cases of metastatic colorectal adenocarcinoma in regional lymph nodes (n=71) and liver (n=36). Results The cases of lymph-node metastasis also had tissues from the primary carcinoma (n=71) and matching normal non-neoplastic mucosal tissues (n=71) from the colon and rectum available for the study. More than half of the cases of primary colorectal carcinoma (43/71; 60%) displayed strong nm23 immunoreactivity, with a similar proportion of the lymph-node metastases (40/71 cases; 56%) having strong nm23 immunostaining. However, only a small minority of the normal controls of non-neoplastic colorectal epithelia (12/71 cases; 17%) had strong nm23 immunoreactivity. The difference in nm23 protein expression between normal colorectal mucosa and primary colorectal carcinoma was statistically significant (P=0.0001; chi-squared test with continuity correction). However, no significant difference in nm23 protein expression was found between primary colorectal carcinoma and lymph-node metastases (P=0.81; chi-squared test with continuity correction). Most of the liver metastases (24/36 cases; 67%) had strong nm23 immunostaining but this finding was not statistically significant when compared with that seen in primary colorectal carcinoma (P=0.62; chi-squared test with continuity correction). In addition, nm23 expression was not found to significantly correlate with 5-year survival of patients with liver metastasis (P=0.86), suggesting that it had no predictive value for overall patient survival. There was also no significant correlation between disease recurrence and nm23 expression (P=0.63).Conclusions In summary, increased nm23 protein immunoreactivity is seen in the majority of colorectal carcinomas when compared to normal colorectal tissues but no significant difference in nm23 expression was found between primary colorectal carcinoma and metastatic carcinoma in regional lymph nodes or the liver. This study suggests that increased nm23 expression may be important in early colorectal carcinoma but not in later progression and dissemination of the tumour. In conclusion, the role and importance of the nm23 gene in the development of tumour metastasis in colorectal carcinoma is questionable.

Increased Expression of nm23 Protein in Colorectal Adenocarcinoma

Some studies have shown that abnormalities of the nm23 gene or its expression may be important in tumor dissemination, suggesting that the gene may have metastasis suppressing activity. The purpose of this study was to determine if the level of nm23 protein expression is altered with progression and dissemination in colorectal adenocarcinoma. Using immunohistochemistry and a monoclonal antibody, NCL-nm23, nm23 protein expression was examined in human primary colorectal adenocarcinoma (n = 100), some with lymph node metastasis (n = 71) and matching normal non-neoplastic colorectal mucosa (n = 100). We found that nm23 protein expression was significantly different between normal colorectal mucosa and primary colorectal carcinoma (p = 0.0001: χ2 test with continuity correction). Almost half of the cases of primary colorectal carcinoma (47/100; 47%) and a similar proportion of the lymphnode metastases (38/71 cases; 54%) had strong nm23 immunostaining. In contrast, only a minority of the normal colorectal mucosal tissues (9/100 cases; 9%) showed strong nm23 expression. However, no significant difference was found between primary colorectal carcinoma and lymph node metastases. Intracase comparisons revealed that the majority of cases (84/100; 84%) had more nm23 immunoreactivity in the primary carcinoma tissues than in the normal mucosa. Reduced nm23 immunoreactivity in the metastasis, when compared to the primary tumor tissues, was found in less than half of the cases (29/71; 41%). In summary, nm23 protein immunoreactivity appears increased in colorectal carcinoma compared to normal colorectal tissues, but no significant difference was seen in nm23 expression between primary colorectal carcinoma and metastatic carcinoma tissues. This suggests that increased nm23 expression may be important in early colorectal carcinoma but not necessarily in later progression or dissemination of the tumor. A large proportion of metastatic carcinomas paradoxically had increased nm23 protein immunoreactivity, contradicting its postulated metastasis-suppressing role. (The J Histotechnol 23:133, 2000)

Increased Expression of nm23 Protein in Colorectal Adenocarcinoma

Some studies have shown that abnormalities of the nm23 gene or its expression may be important in tumor dissemination, suggesting that the gene may have metastasis suppressing activity. The purpose of this study was to determine if the level of nm23 protein expression is altered with progression and dissemination in colorectal adenocarcinoma. Using immunohistochemistry and a monoclonal antibody, NCL-nm23, nm23 protein expression was examined in human primary colorectal adenocarcinoma (n = 100), some with lymph node metastasis (n = 71) and matching normal non-neoplastic colorectal mucosa (n = 100). We found that nm23 protein expression was significantly different between normal colorectal mucosa and primary colorectal carcinoma (p = 0.0001: χ2 test with continuity correction). Almost half of the cases of primary colorectal carcinoma (47/100; 47%) and a similar proportion of the lymphnode metastases (38/71 cases; 54%) had strong nm23 immunostaining. In contrast, only a minority of the normal colorectal mucosal tissues (9/100 cases; 9%) showed strong nm23 expression. However, no significant difference was found between primary colorectal carcinoma and lymph node metastases. Intracase comparisons revealed that the majority of cases (84/100; 84%) had more nm23 immunoreactivity in the primary carcinoma tissues than in the normal mucosa. Reduced nm23 immunoreactivity in the metastasis, when compared to the primary tumor tissues, was found in less than half of the cases (29/71; 41%). In summary, nm23 protein immunoreactivity appears increased in colorectal carcinoma compared to normal colorectal tissues, but no significant difference was seen in nm23 expression between primary colorectal carcinoma and metastatic carcinoma tissues. This suggests that increased nm23 expression may be important in early colorectal carcinoma but not necessarily in later progression or dissemination of the tumor. A large proportion of metastatic carcinomas paradoxically had increased nm23 protein immunoreactivity, contradicting its postulated metastasis-suppressing role. (The J Histotechnol 23:133, 2000)

ＲＧＤ構造類似体アルギニルアミノヘキサン酸およびその修飾リポソームによる癌転移抑制

Arg-Gly-Asp (RGD) amino acid sequence was originally found in fibronectin, a main component of extracellular matrices, and is known as a ligand to some adhesion molecules. Synthetic peptides based on the sequence have been found to decrease metastatic colonization of metastatic tumors. To attempt stabilization and prolong the circulation time of a compound having RGD sequence, an RGD mimetic (arginyl-aminohexanoic acid, NOK) and its hydrophobized derivative (NOK-L) were synthesized. At first, the effect of NOK and liposomal NOK-L on adhesion properties of tumor cells in vitro was investigated. NOK inhibited adhesion of B 16 BL 6 murine melanoma cells to immobilized NOK-L. Liposomal NOK-L inhibited adhesion of the cells to fibronectin, suggesting that NOR mimic the function of RGD, Next, we investigated the metastasis-suppressing efficacy of NOK and liposomal NOK-L in vivo, using the models of both experimental and spontaneous metastasis of B 16 BL 6 cells. As a result, a significant decrease in lung colonization was observed in a group injected with liposomal NOK-L in both models. These results indicate that NOR has the metastasis-suppressing activity via the mechanism similar to that by RGD, and that liposomalization of NOK using NOK-L augmented the metastasis-suppressing activity. Thus, liposomal NOK may be useful as an antimetastatic agent.