Proteomics-based strategy to delineate the molecular mechanisms of RhoGDI2-induced metastasis and drug resistance in gastric cancer

Rho GDP dissociation inhibitor 2 (RhoGDI2) was initially identified as a regulator of the Rho family of GTPases. Our recent works suggest that RhoGDI2 promotes tumor growth and malignant progression, as well as enhances chemoresistance in gastric cancer. Here, we delineate the mechanism by which RhoGDI2 promotes gastric cancer cell invasion and chemoresistance using two-dimensional gel electrophoresis (2-DE) on proteins derived from a RhoGDI2-overexpressing SNU-484 human gastric cancer cell line and control cells. Differentially expressed proteins were identified using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). In total, 47 differential protein spots were identified; 33 were upregulated, and 14 were downregulated by RhoGDI2 overexpression. Upregulation of SAE1, Cathepsin D, Cofilin1, CIAPIN1, and PAK2 proteins was validated by Western blot analysis. Loss-of-function analysis using small interference RNA (siRNA) directed against candidate genes reveals the need for CIAPIN1 and PAK2 in RhoGDI2-induced cancer cell invasion and Cathepsin D and PAK2 in RhoGDI2-mediated chemoresistance in gastric cancer cells. These data extend our understanding of the genes that act downstream of RhoGDI2 during the progression of gastric cancer and the acquisition of chemoresistance.     

Suppression of human melanoma metastasis by the metastasis suppressor gene, BRMS1

We recently identified a novel metastasis suppressor gene, BRMS1, in breast cancer. Since the BRMS1 gene maps to chromosome 11q13.1-q13.2 and since chromosome 11q defects have been described in various stages of human melanoma progression, we hypothesized that BRMS1 may function as a tumor or metastasis suppressor in melanomas as well. Quantitative real-time RT-PCR revealed that BRMS1 mRNA expression was high in melanocytes, considerably reduced in early melanoma-derived cell lines, and barely detectable in advanced/metastatic cell lines. Stable transfectants of BRMS1 in the human melanoma cell lines MelJuSo and C8161.9 did not alter the tumorigenicity of either cell line, but significantly suppressed metastasis compared to vector-only transfectants. Orthotopic tumors continued to express BRMS1, but expression was lost in lung metastases. In vitro morphology, growth rate, and histology of BRMS1 transfectants were similar to controls. BRMS1 transfectants were less invasive in a collagen sandwich assay and had restored homotypic gap junctional intercellular communication (GJIC). Thus, BRMS1 functions as a metastasis suppressor in more than one tumor type (i.e., breast carcinoma and cutaneous melanoma) by modifying several metastasis-associated phenotypes.     

肿瘤转移抑制基因BRMS1的表达分析及机制研究进展

乳腺癌转移抑制基因1(BRMS1)是一个有活性的肿瘤转移抑制基因,参与抑制乳腺癌、黑素瘤、鼻咽癌、非小细胞肺癌、卵巢癌等恶性肿瘤的转移。BRMS1编码蛋白主要通过转录调控转移相关靶基因,参与调节细胞凋亡、细胞通讯、肿瘤血管新生等多种细胞事件。从BRMS1基因的分子结构、表达调控、生物学功能以及转移抑制机理等方面对BRMS1的研究进展做简要回顾。     

Modelling and simulation of mutant alleles of breast cancer metastasis suppressor 1 (BRMS1) gene

Computational tools occupy the prime position in the analysis of large volume of post-genomic data. These tools have advantage over the wet lab experiments in terms of high coverage, cost and time. Breast cancer is the most common cancer in females worldwide. It is a genetically heterogeneous disorder and many genes are involved in the pathway of the disease. Mutations in metastasis suppressor gene are the major cause of the disease. In this study, the effects of mutations in breast cancer metastasis suppressor 1gene upon protein structure and function were examined by means of computational tools and information from databases.This study can be useful to predict the potential effect of every allelic variant, devise new biological experiments and to interpret and predict the patho-physiological impact of new mutations or non-synonymous polymorphisms.     

Purification and characterisation of the breast cancer metastasis suppressor, BRMS1

The breast cancer metastasis suppressor 1 (BRMS1) is a member of a family of proteins that actively suppress tumour metastasis. Understanding BRMS1 mediated metastasis suppression is critical to the development of new therapies designed to prevent and treat patients with late stage breast cancer. To aid research into the functional aspects that underpin BRMS1 mediated metastasis suppression we have expressed and purified recombinant BRMS1 and produced BRMS1 polyclonal antibodies. Using these antibodies to immunoprecipitate endogenous BRMS1 containing complexes from MCF7 breast cancer cell lines we have identified, by mass spectrometry, the small heat shock protein Hsp27 in complex with BRMS1. We also show that the expression of both BRMS1 and Hsp27 are inversely correlated with metastatic potential.     

Breast cancer metastasis suppressor 1 (BRMS1) is destabilized by the Cul3-SPOP E3 ubiquitin ligase complex

Breast cancer metastasis suppressor 1 (BRMS1) suppresses metastasis without affecting primary tumorigenesis. The regulatory mechanism of BRMS1 at the protein level has not been revealed until recently. Here, we found that cullin 3 (Cul3), a component of E3 ubiquitin ligase, is a new binding partner of BRMS1 and the interaction between BRMS1 and Cul3 is mediated by the SPOP adaptor protein. Intriguingly, BRMS1 turns out to be a potent substrate that is ubiquitinated by the Cul3–SPOP complex. Knockdown of SPOP increases the level of BRMS1 protein and represses the expression of BRMS1 repressive target genes such as OPN and uPA in breast cancer cells. These results suggest that the novel regulatory mechanism of BRMS1 by Cul3–SPOP complex is important for breast cancer progression.     

Two mechanisms involving the autophagic and proteasomal pathways process the metastasis suppressor protein,N-myc downstream regulated gene 1

N-myc downstream regulated gene 1 (NDRG1) is an intriguing metastasis suppressor protein, which plays an important role in suppressing multiple oncogenic signaling pathways. Interestingly, multiple isoforms of NDRG1 have been identified, although the molecular mechanisms involved in their generation remains elusive. Herein, we demonstrate the role of two mechanisms involving autophagic and proteasomal machinery as part of an intricate system to generate different NDRG1 isoforms. Examining multiple pancreatic cancer cell-types using immunoblotting demonstrated three major isoforms of NDRG1 at approximately 41-, 46- and 47-kDa. The top NDRG1 band at 47-kDa was shown to be processed by the proteasome, followed by autophagic metabolism of the middle NDRG1 band at 46-kDa. The role of the proteasomal and autophagic pathways in NDRG1 processing was further confirmed by co-localization analysis of confocal images using PSMD9 and LC3 as classical markers of these respective pathways. All NDRG1 isoforms were demonstrated to be, at least in part, phosphorylated forms of the protein. Inhibition of two well-characterized upstream kinases of NDRG1, namely GSK3β and SGK1, resulted in decreased levels of the top NDRG1 band. Studies demonstrated that inhibition of GSK3β decreased levels of the top 47-kDa NDRG1 band, independent of its kinase activity, and this effect was not mediated via the proteasomal pathway. In contrast, the decrease in the top NDRG1 band at 47-kDa after SGK1 inhibition, was due to suppression of its kinase activity. Overall, these studies elucidated the complex and intricate regulatory pathways involving both proteasomal and autophagic processing of the metastasis suppressor protein, NDRG1.     

The molecular mechanisms that underlie the tumor suppressor function of LKB1

Germline mutations of the LKB1 tumor suppressor gene result in Peutz-Jeghers syndrome （PJS） characterized by intestinal hamartomas and increased incidence of epithelial cancers. Inactivating mutations in LKB1 have also been found in certain sporadic human cancers and with particularly high frequency in lung cancer. LKB1 has now been demonstrated to play a crucial role in pulmonary tumorigenesis, controlling initiation, differentiation, and metastasis. Recent evidences showed that LKB1 is a multitasking kinase, with great potential in orchestrating cell activity. Thus far, LKB1 has been found to play a role in cell polarity, energy metabolism, apoptosis, cell cycle arrest, and cell proliferation, all of which may require the tumor suppressor function of this kinase and/or its catalytic activity. This review focuses on remarkable recent findings concerning the molecular mechanism by which the LKB1 protein kinase operates as a tumor suppressor and discusses the rational treatment strategies to individuals suffering from PJS and other common disorders related to LKB1 signaling.     

Dynamics of metastasis suppressor gene inactivation

For most cancer cell types, the acquisition of metastatic ability leads to clinically incurable disease. Twelve metastasis suppressor genes (MSGs) have been identified that reduce the metastatic propensity of cancer cells. If these genes are inactivated in both alleles, metastatic ability is promoted. Here, we develop a mathematical model of the dynamics of MSG inactivation and calculate the expected number of metastases formed by a tumor. We analyse the effects of increased mutation rates and different fitness values of cells with one or two inactivated alleles on the ability of a tumor to form metastases. We find that mutations that are negatively selected in the main tumor are unlikely to be responsible for the majority of metastases produced by a tumor. Most metastases-causing mutations will be present in all (or most) cells in the main tumor.     

The role of the metastasis suppressor gene KAI1 in melanoma angiogenesis

The tetraspan protein KAI1 (CD82) has been previously shown to have important roles in cell migration, invasion, and melanoma prognosis. In this study, we investigated the role of KAI1 regarding melanoma angiogenesis. KAI1 overexpression strongly suppressed the growth of the human umbilical vein endothelial cells and their tubular structure formation in vitro. Also, KAI1 was able to inhibit both interleukin‐6 (IL‐6) and VEGF at mRNA and protein levels. Using nude mice in the in vivo study, we showed that KAI1, through the regulation of ING4, inhibited blood vessel formation in matrigel plugs along with the downregulation of IL‐6 and VEGF, and the recruitment of CD31‐positive cells. Finally, we found that KAI1 was able to suppress the activity of a serine/threonine kinase Akt by suppressing Akt phosphorylation (Ser473). Taken together, our results suggested that KAI1 was able to suppress melanoma angiogenesis by downregulating IL‐6 and VEGF expression, and the restoration of KAI1 functionality offered a new approach in human melanoma treatment.     

The iron-regulated metastasis suppressor, Ndrg-1: identification of novel molecular targets

A recently identified metastasis suppressor, N-myc downstream regulated gene-1 (Ndrg-1), has been shown to reduce the invasion and metastasis of breast, colon, prostate and pancreatic cancer. Among its many functions, Ndrg-1 is involved in modulating differentiation, proliferation and angiogenesis. However, knowledge of the molecular targets of Ndrg-1 is limited. The current study has focused on examining the functions of Ndrg-1 in a number of different cancer cell models including prostate, colon, lung and pancreatic cancer to elucidate the known pleiotropic nature of this protein. Furthermore, the potential gene targets of Ndrg-1 were analyzed using whole genome gene array revealing a substantial number of genes whose expression was affected by this metastasis suppressor. Significantly, Ndrg-1 up-regulated thiamine triphosphatase (Thtpa) expression in three of the four cell models. Thtpa is known to decrease the levels of the energy currency molecule, thiamine triphosphate, suggesting a potential pathway for the anti-proliferative effects of Ndrg-1. Furthermore, Ndrg-1 reduced the protein levels of cathepsin C which plays a role in invasion, indicating a potential mechanism of its anti-metastatic role in pancreatic cancer cells. These findings provide a potential link between the observed functions of Ndrg-1 and its molecular targets, further demonstrating its anti-metastatic effect.     

Clinical-translational strategies for the elevation of Nm23-H1 metastasis suppressor gene expression

Interruption of the tumor metastatic process is a new, thought provoking molecular target for the treatment of cancer. The Nm23-H1 metastasis suppressor gene stands as a validated molecular target owing to its reduced expression in many aggressive human tumors, and the reduction in metastatic potential in vivo upon re-expression in multiple cell lines. Several compounds have been identified which elevate Nm23-H1 expression in vitro including indomethacin, γ Linolenic Acid, trichostatin A, 5-aza-deoxycytidine, and high dose medroxyprogesterone acetate. Using a model of lung metastatic colonization by MDA-MB-231 human breast carcinoma cells, we demonstrated that high dose MPA reduced the formation of overt lung metastases by 37–46% and those metastases that formed were statistically smaller. A Phase II clinical trial of high dose MPA, alone or in combination with metronomic chemotherapy has recently opened.     

KAI1 tetraspanin and metastasis suppressor

KAI1 is a widely expressed transmembrane glycoprotein of the tetraspanin family. Substantial experimental evidence suggests that KAI1 is an important regulator of cell behaviour. A loss of KAI1 expression is also associated with the advanced stages of many human malignancies and results in the acquisition of invasive and metastatic capabilities by tumour cells, yet the underlying mechanisms responsible for this down-regulation of KAI1 expression remain to be resolved. The recent identification of signalling pathways downstream of KAI1, and proteins that specifically interact with KAI1, are beginning to elucidate the biological pathways involving KAI1.