A tumor suppressor DLC1: The functions and signal pathways

Deleted in liver cancer-1 (DLC1), a potential tumor suppressor, acts as a GTPase-activating protein for Rho family members. In many human cancers, the DLC1 expression is frequently downregulated or inactivated, which allows cancer cells to proliferate and disseminate. In this review, we describe the characteristics and other members of the DLC1 family and delineate the signal pathways DLC1 involved in regulating cancer cell growth, colony formation, apoptosis, senescence, autophagy, migration and invasion. In addition, we explore the clinical data of DLC1 and the mechanisms that natural products upregulate the DLC1 expression to inhibit cancer. Despite these insights, many important unanswered questions remain about the exact mechanisms of DLC1-mediated cancer suppression.     

Analysis of interactions between mismatch repair initiation factors and the replication processivity factor PCNA

In eukaryotes, the DNA replication factor PCNA is loaded onto primer-template junctions to act as a processivity factor for DNA polymerases. Genetic and biochemical studies suggest that PCNA also functions in early steps in mismatch repair (MMR) to facilitate the repair of misincorporation errors generated during DNA replication. These studies have shown that PCNA interacts directly with several MMR components, including MSH3, MSH6, MLH1, and EXO1. At present, little is known about how these interactions contribute to the mismatch repair mechanism. The interaction between MLH1 and PCNA is of particular interest because MLH1-PMS1 is thought to act as a matchmaker to signal mismatch recognition to downstream repair events; in addition, PCNA has been hypothesized to act in strand discrimination steps in MMR. Here, we utilized both genetic and surface plasmon resonance techniques to characterize the MLH1-PMS1-PCNA interaction. These analyses enabled us to determine the stability of the complex (K(D) = 300 nM) and to identify residues (572-579) in MLH1 and PCNA (126,128) that appear important to maintain this stability. We favor a model in which PCNA acts as a scaffold for consecutive protein-protein interactions that allow for the coordination of MMR steps.     

MicroRNA-487a-3p functions as a new tumor suppressor in prostate cancer by targeting CCND1

Prostate cancer (PCa) is one of the major health problems of the aging male. The roles of dysregulated microRNAs in PCa remain unclear. In this study, we mined the public published data and found that miR-487a-3p was significantly downregulated in 38 pairs of clinical prostate tumor tissues compared with the normal tissues. We further verified this result by in situ hybridization on tissue chip and quantitative real-time polymerase chain reaction (qRT-PCR) in PCa/normal cells. miR-487a-3p targeting of cyclin D1 (CCND1) was identified using bioinformatics, qRT-PCR and western blot analyses. The cellular proliferation, cell cycle, migration, and invasion were assessed by cell counting kit-8, flow cytometry analysis and transwell assay. We discovered that overexpression of miR-487a-3p suppressed PCa cell growth, migration, invasion by directly targeting CCND1. Knockdown of CCND1 in PCa cells showed similar results. Meanwhile, the expression level of CCND1 was significantly upregulated in the PCa tissues and cell lines, which presented negative correlation with the expression of miR-487a-3p. More important, we demonstrated significantly reduced growth of xenograft tumors of stable miR-487a-3p-overexpressed human PCa cells in nude mice. Taken together, for the first time, our results revealed that miR-487a-3p as a tumor suppressor of PCa could target CCND1. Our finding might reveal miR-487a-3p could be potentially contributed to the pathogenesis and a clinical biomarker or the new potential therapeutic target of PCa.     

Msh2 separation of function mutations confer defects in the initiation steps of mismatch repair

In eukaryotes the MSH2-MSH3 and MSH2-MSH6 heterodimers initiate mismatch repair (MMR) by recognizing and binding to DNA mismatches. The MLH1-PMS1 heterodimer then interacts with the MSH proteins at or near the mismatch site and is thought to act as a mediator to recruit downstream repair proteins. Here we analyzed five msh2 mutants that are functional in removing 3' non-homologous tails during double-strand break repair but are completely defective in MMR. Because non-homologous tail removal does not require MSH6, MLH1, or PMS1 functions, a characterization of the msh2 separation of function alleles should provide insights into early steps in MMR. Using the Taq MutS crystal structure as a model, three of the msh2 mutations, msh2-S561P, msh2-K564E, msh2-G566D, were found to map to a domain in MutS involved in stabilizing mismatch binding. Gel mobility shift and DNase I footprinting assays showed that two of these mutations conferred strong defects on MSH2-MSH6 mismatch binding. The other two mutations, msh2-S656P and msh2-R730W, mapped to the ATPase domain. DNase I footprinting, ATP hydrolysis, ATP binding, and MLH1-PMS1 interaction assays indicated that the msh2-S656P mutation caused defects in ATP-dependent dissociation of MSH2-MSH6 from mismatch DNA and in interactions between MSH2-MSH6 and MLH1-PMS1. In contrast, the msh2-R730W mutation disrupted MSH2-MSH6 ATPase activity but did not strongly affect ATP binding or interactions with MLH1-PMS1. These results support a model in which MMR can be dissected into discrete steps: stable mismatch binding and sensing, MLH1-PMS1 recruitment, and recycling of MMR components.     

LKB1 in lung cancerigenesis: a serine/threonine kinase as tumor suppressor

Lung cancer is featured with high mortality, with a 15% five-year survival rate worldwide. Genetic alterations, such as loss of function of tumor suppressor genes, frequently contribute to lung cancer initiation, progression and metastasis. Liver kinase B1 (LKB1), as a serine/threonine kinase and tumor suppressor, is frequently mutated and inactivated in non-small cell lung cancer (NSCLC). Recent studies have provided strong evidences that LKB1 loss promotes lung cancerigenesis process, especially lung cancer progression and metastasis. This review will summarize recent progress on how LKB1 modulates the process of lung cancerigenesis, emphasizing on LKB1 downstream signaling pathways and biological functions. We will further discuss the potential development of prognostic biomarkers or therapeutic targets in lung cancer clinic based on the molecular alteration associated with deregulated LKB1 signaling.     

Crosstalk between BRCA‐Fanconi anemia and mismatch repair pathways prevents MSH2‐dependent aberrant DNA damage responses

Several proteins in the BRCA‐Fanconi anemia (FA) pathway, such as FANCJ, BRCA1, and FANCD2, interact with mismatch repair (MMR) pathway factors, but the significance of this link remains unknown. Unlike the BRCA‐FA pathway, the MMR pathway is not essential for cells to survive toxic DNA interstrand crosslinks (ICLs), although MMR proteins bind ICLs and other DNA structures that form at stalled replication forks. We hypothesized that MMR proteins corrupt ICL repair in cells that lack crosstalk between BRCA‐FA and MMR pathways. Here, we show that ICL sensitivity of cells lacking the interaction between FANCJ and the MMR protein MLH1 is suppressed by depletion of the upstream mismatch recognition factor MSH2. MSH2 depletion suppresses an aberrant DNA damage response, restores cell cycle progression, and promotes ICL resistance through a Rad18‐dependent mechanism. MSH2 depletion also suppresses ICL sensitivity in cells deficient for BRCA1 or FANCD2, but not FANCA. Rescue by Msh2 loss was confirmed in Fancd2‐null primary mouse cells. Thus, we propose that regulation of MSH2‐dependent DNA damage response underlies the importance of interactions between BRCA‐FA and MMR pathways.     

P42 Ebp1 functions as a tumor suppressor in non-small cell lung cancer

Although the short isoform of ErbB3-binding protein 1 (Ebp1), p42 has been considered to be a potent tumor suppressor in a number of human cancers, whether p42 suppresses tumorigenesis of lung cancer cells has never been clarified. In the current study we investigated the tumor suppressor role of p42 in non-small cell lung cancer cells. Our data suggest that the expression level of p42 is inversely correlated with the cancerous properties of NSCLC cells and that ectopic expression of p42 is sufficient to inhibit cell proliferation, anchorage-independent growth, and invasion as well as tumor growth in vivo. Interestingly, p42 suppresses Akt activation and overexpression of a constitutively active form of Akt restores the tumorigenic activity of A549 cells that is ablated by exogenous p42 expression. Thus, we propose that p42 Ebp1 functions as a potent tumor suppressor of NSCLC through interruption of Akt signaling. [BMB Reports 2015; 48(3): 159-165]     

Nit1 and Fhit tumor suppressor activities are additive

The fragile histidine triad gene (human FHIT, mouse Fhit) has been shown to act as a tumor suppressor gene. Nit1 and Fhit form a fusion protein, encoded by the NitFhit gene in flies and worms, suggesting that mammalian Nit1 and Fhit proteins, which are encoded by genes on different chromosomes in mammals, may function in the same signal pathway(s). A previous study showed that Nit1 deficiency in knockout mice confers a cancer prone phenotype, as does Fhit deficiency. We have now assessed the tumor susceptibility of Fhit^?/?Nit1^?/? mice and observed that double knockout mice develop more spontaneous and carcinogen‐induced tumors than Fhit^?/? mice, suggesting that the extent of tumor susceptibility due to Nit1 and Fhit deficiency is additive, and that Nit1 and Fhit affect distinct signal pathways in mammals. Nit1, like Fhit, is present in cytoplasm and mitochondria but not nuclei. Because Fhit deficiency affects responses to replicative and oxidative stress, we sought evidence for Nit1 function in response to such stresses in tissues and cultured cells: when treated with hydroxyurea, the normal kidney‐derived double‐deficient cells appear not to activate the pChk2 pathway and when treated with H₂O₂, show little evidence of DNA damage, compared with wild type and Fhit^?/? cells. The relevance of Nit1 deficiency to human cancers was examined in human esophageal cancer tissues, and loss of Nit1 expression was observed in 48% of esophageal adenocarcinomas. J. Cell. Biochem. 107: 1097–1106, 2009. © 2009 Wiley‐Liss, Inc.     

The tumor suppressor BAP1 cooperates with BRAFV600E to promote tumor formation in cutaneous melanoma

The deubiquitinating enzyme BAP1 is mutated in a hereditary cancer syndrome with a high risk of mesothelioma and melanocytic tumors. Here, we show that Bap1 deletion in melanocytes cooperates with the constitutively active, oncogenic form of BRAF (BRAF^V600E) and UV to cause melanoma in mice, albeit at very low frequency. In addition, Bap1‐null melanoma cells derived from mouse tumors are more aggressive and colonize and grow at distant sites more than their wild‐type counterparts. Molecularly, Bap1‐null melanoma cell lines have increased DNA damage measured by γH2aX and hyperubiquitination of histone H2a. Therapeutically, these Bap1‐null tumors are completely responsive to BRAF‐ and MEK‐targeted therapies. Therefore, BAP1 functions as a tumor suppressor and limits tumor progression in melanoma.     

RASSF tumor suppressor gene family: Biological functions and regulation

Genetic changes through allelic loss and nucleic acid or protein modifications are the main contributors to loss of function of tumor suppressor proteins. In particular, epigenetic silencing of genes by promoter hypermethylation is associated with increased tumor severity and poor survival. The RASSF (Ras association domain family) family of proteins consists of 10 members, many of which are tumor suppressor proteins that undergo loss of expression through promoter methylation in numerous types of cancers such as leukemia, melanoma, breast, prostate, neck, lung, brain, colorectal and kidney cancers. In addition to their tumor suppressor function, RASSF proteins act as scaffolding agents in microtubule stability, regulate mitotic cell division, modulate apoptosis, control cell migration and cell adhesion, and modulate NFκB activity and the duration of inflammation. The ubiquitous functions of these proteins highlight their importance in numerous physiological pathways. In this review, we will focus on the biological roles of the RASSF family members and their regulation.     

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。     

Expression and clinicopathological significance of lipin-1 in human breast cancer and its association with p53 tumor suppressor gene

Breast cancer (BC) is an important cause of female cancer-related death. It has recently been demonstrated that metabolic disorders including lipid metabolism are a hallmark of cancer cells. Lipin-1 is an enzyme that displays phosphatidate phosphatase activity and regulates the rate-limiting step in the pathway of triglycerides and phospholipids synthesis. The objective of this study was to evaluate lipin-1 expression, its prognostic significance, and its correlation with p53 tumor suppressor in patients with BC. In this study, 55 pairs of fresh samples of BC and adjacent noncancerous tissue were used to analyze lipin-1, using quantitative real-time polymerase chain reaction and immunohistochemistry (IHC) staining. The expression of other clinicopathological variables and p53 was also examined using IHC technique. The cell migration was studied in MCF-7 and MDA-MB231 cells following the inhibition of lipin-1 by propranolol. Our results show that the relative expression of lipin-1 messenger RNA was significantly higher in BC tissues compared with the adjacent normal tissue and its inhibition reduced cell migration in cancer cells. This upregulation was negatively correlated with histological grade of tumor and p53 status (p = .001 and p = .034) respectively and positively correlated with the tumor size (p = .006). Our results also seem to indicate that the high lipin-1 expression is related to a good prognosis in patients with BC. The expression of lipin-1 may be considered as a novel independent prognostic factor. The inhibition of lipin-1 may also have therapeutic significance for patients with BC. The correlation between lipin-1 and p53 confirms the role of p53 in the regulation of lipid metabolism in cancer cells.     

Effects of 2-amino-4,6-dinitrotoluene on p53 tumor suppressor gene expression

2-Amino-4,6-dinitrotoluene (2-Am-DNT) and its isomers are the most common metabolites of 2,4,6-trinitrotoluene (TNT). It is desirable to know the toxicity of this metabolite particularly because of its role in carcinogenicity and because it could contaminate drinking water. We used MCF-7 human breast cancer cells which have p53 tumor suppressor gene in wild type form in both the loci. Immunoblotting with p53 antibody showed enhanced p53 level in treated cells compared to untreated control cells. Similarly, p53 DNA-protein binding assays (gel-shift) showed accumulation of p53 protein in treated cells. This is the first report which shows p53 accumulation in 2-Am-DNT treated cells providing evidence of potential carcinogenic effects of 2-Am-DNT.     

Hoxc8 downregulates Mgl1 tumor suppressor gene expression and reduces its concomitant function on cell adhesion

Hoxc8 is a homeobox gene family member, which is essential for growth and differentiation. Mgl1, a mouse homologue of the Drosophila tumor suppressor gene lgl, was previously identified as a possible target of Hoxc8. However, the biological effects and underlying molecular mechanism of Hoxc8 regulation on Mgl1 has not been fully established. The endogenous expression patterns of Hoxc8 were inversely correlated with those of Mgl1 in different types of cells and tissues. Here we showed that Hoxc8 overexpression downregulated the Mgl1 mRNA expression. Characterization of the ∼2 kb Mgl1 promoter region revealed that the upstream sequence contains several putative Hox core binding sites and chromatin immunoprecipitation assay confirmed that Hoxc8 directly binds to the 5′ upstream region of Mgl1. The promoter activity of this region was diminished by Hoxc8 expression but resumed by knockdown of Hoxc8 using siRNA against Hoxc8. Functional study of Mgl1 in C3H10T1/2 cells revealed a significant reduction in cell adhesion upon expression of Hoxc8. Taken together, our data suggest that Hoxc8 downregulates Mgl1 expression via direct binding to the promoter region, which in turn reduces cell adhesion and concomitant cell migration.     

Construction of a dual affinity tagged allele of the Rb1 tumor suppressor gene in the mouse

Loss of Rb1 tumor suppressor gene function is involved in the genesis of most human cancers. Novel therapies targeting Rb1 have been slow to develop because of our incomplete understanding of its molecular mechanisms of action. Rb1 protein (pRb) binds a host of cellular genes and proteins, and these molecular interactions mediate its various functions. Given the potential complexity of these molecular interactions and the lack of established methods for pRb purification, it has been difficult to systematically identify gene and protein interactions relevant to tumor suppression in different tissues in vivo. To address this limitation, we have generated a dual affinity tagged Rb1 allele in the mouse. The tagged allele functions as wild type and the encoded protein can be purified by tandem affinity chromatography. This allele will facilitate identification and characterization of native pRb molecular interactions in any tissue accessible in the mouse. genesis 48:121–126, 2010. © 2009 Wiley‐Liss, Inc.     

Generation of two modified mouse alleles of the Hic1 tumor suppressor gene

HIC1 (hypermethylated in cancer 1) is a tumor suppressor gene located on chromosome 17p13.3, a region frequently hypermethylated or deleted in human neoplasias. In mouse, Hic1 is essential for embryonic development and exerts an antitumor role in adult animals. Since Hic1-deficient mice die perinatally, we generated a conditional Hic1 null allele by flanking the Hic1-coding region by loxP sites. When crossed to animals expressing Cre recombinase in a cell-specific manner, the Hic1 conditional mice will provide new insights into the function of Hic1 in developing and mature tissues. Additionally, we used gene targeting to replace sequence-encoding amino acids 186-893 of Hic1 by citrine fluorescent protein cDNA. We demonstrate that the distribution of Hic1-citrine fusion polypeptide corresponds to the expression pattern of wild-type Hic1. Consequently, Hic1-citrine "reporter" mice can be used to monitor the activity of the Hic1 locus using citrine fluorescence.     

Thioredoxin interacting protein (TXNIP) acts as a tumor suppressor in human prostate cancer

Prostate cancer (PCa) is one of the most common malignant tumors in the world. Thioredoxin interacting protein (TXNIP) is downregulated in a variety of human tumors and plays an important role in tumor suppression. However, the expression level and biological functions of TXNIP in PCa have not been identified yet. Therefore, this study aims to investigate the expression and biological functions of TXNIP in PCa. We reported that the expression of TXNIP was significantly decreased in PCa and associated with clinicopathological features. Overexpression of TXNIP could significantly inhibited PC‐3 cells proliferation, migration, invasion, and glucose uptake. Additionally, overexpression of TXNIP could remarkably block cell cycle in the G0/G1 phase and promoted cell apoptosis. Furthermore, TXNIP expression correlated inversely with GLUT1 expression in PCa. Taken together, our results for the first time revealed that TXNIP was decreased in PCa. Moreover, TXNIP might act as a tumor suppressor of PCa and correlated with tumor occurrence and development. Our findings cast a new light on better understanding the occurrence and development of PCa and indicated that TXNIP might be favorable for PCa molecular target therapy.     

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.