GRWD1, a new player among oncogenesis-related ribosomal/nucleolar proteins

Increasing attention has been paid to certain ribosomal or ribosome biosynthesis-related proteins involved in oncogenesis. Members of one group are classified as “tumor suppressive factors” represented by RPL5 and RPL11; loss of their functions leads to cancer predisposition. RPL5 and RPL11 prevent tumorigenesis by binding to and inhibiting the MDM2 ubiquitin ligase and thereby up-regulating p53. Many other candidate tumor suppressive ribosomal/nucleolar proteins have been suggested. However, it remains to be experimentally clarified whether many of these factors can actually prevent tumorigenesis and if so, how they do so. Conversely, some ribosomal/nucleolar proteins promote tumorigenesis. For example, PICT1 binds to and anchors RPL11 in nucleoli, down-regulating p53 and promoting tumorigenesis. GRWD1 was recently identified as another such factor. When overexpressed, GRWD1 suppresses p53 and transforms normal human cells, probably by binding to RPL11 and sequestrating it from MDM2. However, other pathways may also be involved.     

SIRT1, Is It a Tumor Promoter or Tumor Suppressor?

SIRT1 has been considered as a tumor promoter because of its increased expression in some types of cancers and its role in inactivating proteins that are involved in tumor suppression and DNA damage repair. However, recent studies demonstrated that SIRT1 levels are reduced in some other types of cancers, and that SIRT1 deficiency results in genetic instability and tumorigenesis, while overexpression of SIRT1 attenuates cancer formation in mice heterozygous for tumor suppressor p53 or APC. Here, I review these recent findings and discuss the possibility that activation of SIRT1 both extends lifespan and inhibits cancer formation.     

The Expression of SIRT1 and DBC1 in Laryngeal and Hypopharyngeal Carcinomas

Rationale and Objective

Sirtuin 1 (SIRT1) plays an important role in tumorigenesis and is increased in many human tumors. DBC1 is a negative regulator of SIRT1 via promotion of p53-mediated apoptosis. It is necessary to investigate the expression of SIRT1 and DBC1 in laryngeal and hypopharyngeal squamous cell carcinomas (LSCC and HSCC) and its correlation with available clinical parameters.

Methods

The mRNA levels of SIRT1 and DBC1 were measured in 54 paired LSCC or HSCC tumors and corresponding adjacent noncancerous mucosae using quantitative RT-PCR (qRT-PCR). The protein levels of SIRT1 and DBC1 were also evaluated in 120 cases of patients with LSCC or HSCC using immunohistochemical staining. The correlation between SIRT1 and DBC1 expression and clinical parameters was analyzed with Pearson chi-square test.

Results

qRT-PCR assay showed that, compared with the paired adjacent noncancerous mucosae, SIRT1 mRNA was significantly decreased in tumors. The immunohistochemical results indicated that the SIRT1 protein was also downregulated in tumors compared with noncancerous mucosae. Moreover, decreased SIRT1 was significantly correlated with the tumor clinical stage and lymph node metastasis. Additionally, DBC1 mRNA was significantly increased in tumors compared with noncancerous mucosae. The immunohistochemical results indicated that the DBC1 protein was downregulated in tumors, which is inconsistent with the results obtained by qRT-PCR. Finally, decreased DBC1 protein was significantly correlated with tumor differentiation, lymph node metastasis, and p53 expression.

Conclusions

SIRT1 and DBC1 might be involved in the pathophysiology of laryngeal and hypopharyngeal squamous cell carcinomas and are associated with lymph node metastasis and p53 positive staining in LSCCs and HSCCs.     

E3B1在肿瘤中的作用及其机制

EPS8的结合蛋白质E3B1具有广泛的生物学功能,参与细胞骨架的重塑、生长因子受体(GFR)介导的细胞应答,抑制细胞生长,并通过E3B1-EPS8-SOS1三联复合物参与Ras到Rac细胞信号转导,这些功能使得E3B1及其家族蛋白质具有潜在的肿瘤抑制作用,与肿瘤的发生、发展密切相关。目前对于E3B1的研究主要集中在其信号通路,对于E3B1在肿瘤中的作用及其机制的研究有待于进一步加强,这些研究可能为肿瘤的转移途径、转化方式的研究提供重要的理论线索。     

Dephosphorylation of DBC1 by Protein Phosphatase 4 Is Important for p53-Mediated Cellular Functions

Deleted in breast cancer-1 (DBC1) contributes to the regulation of cell survival and apoptosis. Recent studies demonstrated that DBC is phosphorylated at Thr454 by ATM/ATR kinases in response to DNA damage, which is a critical event for p53 activation and apoptosis. However, how DBC1 phosphorylation is regulated has not been studied. Here we show that protein phosphatase 4 (PP4) dephosphorylates DBC1, regulating its role in DNA damage response. PP4R2, a regulatory subunit of PP4, mediates the interaction between DBC1 and PP4C, a catalytic subunit. PP4C efficiently dephosphorylates pThr454 on DBC1 in vitro, and the depletion of PP4C/PP4R2 in cells alters the kinetics of DBC1 phosphorylation and p53 activation, and increases apoptosis in response to DNA damage, which are compatible with the expression of the phosphomimetic DBC-1 mutant (T454E). These suggest that the PP4-mediated dephosphorylation of DBC1 is necessary for efficient damage responses in cells.     

Cell cycle checkpoints and their impact on anticancer therapeutic strategies

Cells contain numerous pathways designed to protect them from the genomic instability or toxicity that can result when their DNA is damaged. The p53 tumor suppressor is particularly important for regulating passage through G1 phase of the cell cycle, while other checkpoint regulators are important for arrest in S and G2 phase. Tumor cells often exhibit defects in these checkpoint proteins, which can lead to hypersensitivity; proteins in this class include ataxia-telangiectasia mutatated (ATM), Meiotic recanbination 11 (Mre11), Nijmegen breakage syndrome 1 (Nbs 1), breast cancer susceptibility genes 1 and 2 (BRCA1), and (BRCA2). Consequently, tumors should be assessed for these specific defects, and specific therapy prescribed that has high probability of inducing response. Tumors defective in p53 are frequently considered resistant to apoptosis, yet this defect also provides an opportunity for targeted therapy. When their DNA is damaged, p53-defective tumor cells preferentially arrest in S or G2 phase where they are susceptible to checkpoint inhibitors such as caffeine and UCN-01. These inhibitors preferentially abrogate cell cycle arrest in p53-defective cells, driving them through a lethal mitosis. Wild type p53 can prevent abrogation of arrest by elevating levels of p21(waf1) and decreasing levels of cyclins A and B. During tumorigenesis, tumor cells frequently loose checkpoint controls and this facilitates the development of the tumor. However, these defects also represent an Achilles heel that can be targeted to improve current therapeutic strategies.     

Rap2b promotes proliferation,migration, and invasion of lung cancer cells

Rap2b, a member of the guanosine triphosphate-binding proteins, is widely up-regulated in many types of tumors. However, the functional role of Rap2b in tumorigenesis of lung cancer remains to be fully elucidated. In this study, we investigated the effect of Rap2b on the lung cancer malignant phenotype, such as cell proliferation and metastasis. We found that Rap2b could promote the abilities of lung cancer cell wound healing, migration, and invasion via increasing matrix metalloproteinase-2 enzyme activity. Furthermore, Rap2b overexpression could increase the phosphorylation level of extracellular signal-regulated protein kinases 1/2. In conclusion, our results suggested that Rap2b may be a potential therapeutic target for lung cancer.     

Cyclin D1 down-regulation is essential for DBC2's tumor suppressor function

The expression of tumor suppressor gene DBC2 causes certain breast cancer cells to stop growing [M. Hamaguchi, J.L. Meth, C. Von Klitzing, W. Wei, D. Esposito, L. Rodgers, T. Walsh, P. Welcsh, M.C. King, M.H. Wigler, DBC2, a candidate for a tumor suppressor gene involved in breast cancer, Proc. Natl. Acad. Sci. USA 99 (2002) 13647-13652]. Recently, DBC2 was found to participate in diverse cellular functions such as protein transport, cytoskeleton regulation, apoptosis, and cell cycle control [V. Siripurapu, J.L. Meth, N. Kobayashi, M. Hamaguchi, DBC2 significantly influences cell cycle, apoptosis, cytoskeleton, and membrane trafficking pathways. J. Mol. Biol. 346 (2005) 83-89]. Its tumor suppression mechanism, however, remains unclear. In this paper, we demonstrate that DBC2 suppresses breast cancer proliferation through down-regulation of Cyclin D1 (CCND1). Additionally, the constitutional overexpression of CCND1 prevented the negative impact of DBC2 expression on their growth. Under a CCND1 promoter, the expression of CCNE1 exhibited the same protective effect. Our results indicate that the down-regulation of CCND1 is an essential step for DBC2's growth suppression of cancer cells. We believe that this discovery contributes to a better understanding of DBC2's tumor suppressor function.     

DBC2 resistance is achieved by enhancing 26S proteasome-mediated protein degradation

Tumor suppressor gene DBC2 stops growth of tumor cells through regulation of CCND1. Interference of CCND1 down-regulation prevented growth arrest caused by DBC2 [T. Yoshihara, D. Collado, M. Hamaguchi, Cyclin D1 down-regulation is essential for DBC2's tumor suppressor function, Biochemical and biophysical research communications 358 (2007) 1076-1079]. It was also noted that DBC2 resistant cells eventually arose after repeated induction of DBC2 with muristerone A treatment [M. Hamaguchi, J.L. Meth, C. Von Klitzing, W. Wei, D. Esposito, L. Rodgers, T. Walsh, P. Welcsh, M.C. King, M.H. Wigler, DBC2, a candidate for a tumor suppressor gene involved in breast cancer, Proc. Natl. Acad. Sci. USA 99 (2002) 13647-13652]. In order to elucidate the mechanism of resistance acquisition, we analyzed DBC2 sensitive and resistant cells derived from the same progenitor cells (T-47D). We discovered that DBC2 protein was abundantly expressed in the sensitive cells when DBC2 was induced. In contrast, it was undetectable by western blot analysis in the resistant cells. We confirmed that the inducible gene expression system was responsive in both cells by detecting induced GFP. Additionally, inhibition of 26S proteasome by MG132 revealed production of DBC2 protein in the resistant cells. These findings indicate that the resistant T-47D cells survive DBC2 induction by rapid destruction of DBC2 through 26S proteasome-mediated protein degradation.     

Hypoxia-induced DNp73 stabilization regulates Vegf-A expression and tumor angiogenesis similar to TAp73

P73, the homolog of p53, exists in 2 major forms: either as a pro-apoptotic TAp73 or an amino-terminally truncated DNp73, the latter lacking the first transactivation domain. While TAp73s tumor suppressive functions have been established, DNp73 is an anti-apoptotic protein conferring chemoresistance and is associated with poor survival. However, both forms are variably overexpressed in many human cancers. In this context, we have recently demonstrated that TAp73 is stabilized by hypoxia, a tumor-relevant condition that is associated with cell survival, via HIF-1α-mediated suppression of Siah1 E3 ligase that degrades TAp73. Consequently, hypoxic signals lead to TAp73-mediated activation of several angiogenic genes and blood vessel formation, thereby supporting tumorigenesis. We show here that, similar to TAp73, DNp73 is stabilized by hypoxia in a HIF-1α-dependent manner, which otherwise is degraded by Siah1. Moreover, DNp73 is capable of inducing the expression of Vegf-A, the prototypic angiogenic gene, and loss of DNp73 expression results in reduction in tumor vasculature and size. These data therefore indicate a common mode of regulation for both p73 forms by hypoxia, resulting in the promotion of angiogenesis and tumor growth, highlighting common functionality of these antagonistic proteins under specific physiological contexts.     

Rae1 is an essential mitotic checkpoint regulator that cooperates with Bub3 to prevent chromosome missegregation

The WD-repeat proteins Rae1 and Bub3 show extensive sequence homology, indicative of functional similarity. However, previous studies have suggested that Rae1 is involved in the mRNA export pathway and Bub3 in the mitotic checkpoint. To determine the in vivo roles of Rae1 and Bub3 in mammals, we generated knockout mice that have these genes deleted individually or in combination. Here we show that haplo-insufficiency of either Rae1 or Bub3 results in a similar phenotype involving mitotic checkpoint defects and chromosome missegregation. We also show that overexpression of Rae1 can correct for Rae1 haplo-insufficiency and, surprisingly, Bub3 haplo-insufficiency. Rae1-null and Bub3-null mice are embryonic lethal, although cells from these mice did not have a detectable defect in nuclear export of mRNA. Unlike null mice, compound haplo-insufficient Rae1/Bub3 mice are viable. However, cells from these mice exhibit much greater rates of premature sister chromatid separation and chromosome missegregation than single haplo-insufficient cells. Finally, we show that mice with mitotic checkpoint defects are more susceptible to dimethylbenzanthrene-induced tumorigenesis than wild-type mice. Thus, our data demonstrate a novel function for Rae1 and characterize Rae1 and Bub3 as related proteins with essential, overlapping, and cooperating roles in the mitotic checkpoint.     

Differential Impact of Tumor Suppressor Pathways on DNA Damage Response and Therapy-Induced Transformation in a Mouse Primary Cell Model

The RB and p53 tumor suppressors are mediators of DNA damage response, and compound inactivation of RB and p53 is a common occurrence in human cancers. Surprisingly, their cooperation in DNA damage signaling in relation to tumorigenesis and therapeutic response remains enigmatic. In the context of individuals with heritable retinoblastoma, there is a predilection for secondary tumor development, which has been associated with the use of radiation-therapy to treat the primary tumor. Furthermore, while germline mutations of the p53 gene are critical drivers for cancer predisposition syndromes, it is postulated that extrinsic stresses play a major role in promoting varying tumor spectrums and disease severities. In light of these studies, we examined the tumor suppressor functions of these proteins when challenged by exposure to therapeutic stress. To examine the cooperation of RB and p53 in tumorigenesis, and in response to therapy-induced DNA damage, a combination of genetic deletion and dominant negative strategies was employed. Results indicate that loss/inactivation of RB and p53 is not sufficient for cellular transformation. However, these proteins played distinct roles in response to therapy-induced DNA damage and subsequent tumorigenesis. Specifically, RB status was critical for cellular response to damage and senescence, irrespective of p53 function. Loss of RB resulted in a dramatic evolution of gene expression as a result of alterations in epigenetic programming. Critically, the observed changes in gene expression have been specifically associated with tumorigenesis, and RB-deficient, recurred cells displayed oncogenic characteristics, as well as increased resistance to subsequent challenge with discrete therapeutic agents. Taken together, these findings indicate that tumor suppressor functions of RB and p53 are particularly manifest when challenged by cellular stress. In the face of such challenge, RB is a critical suppressor of tumorigenesis beyond p53, and RB-deficiency could promote significant cellular evolution, ultimately contributing to a more aggressive disease.     

Expression and functional characterization of LRRC4, a novel brain-specific member of the LRR superfamily

LRRC4, a novel member of LRR superfamily thought to be involved in development and tumorigenesis of the nervous tissue, has the potential to suppress tumorigenesis and cell proliferation of U251MG cells. This study aimed at revealing the correlation between expression of LRRC4 and the maintenance of normal function and tumorigenesis suppression within the central nervous system. We systematically analyzed the expression and tissue distributions of the gene in tissues. Results showed that LRRC4 expression was limited to normal adult brain, both in human and in mouse, and exhibited a development-regulated pattern, but was down-regulated in brain tumor tissues and U251MG cell line. Furthermore, dynamic alterations in gene expression associated with cell cycle progression were investigated by using Tet-on system. Results showed that LRRC4 induced a cell cycle delay at the late G1 phase, probably through the alteration of the expression of different cell cycle regulating proteins responsible for mediating G1-S progression, such as p21(Waf1/Cip1) and p27(Kip1), Cdk2 and PCNA, p-ERK1/2. These findings suggest that LRRC4 may play an important role in maintaining normal function and suppressing tumorigenesis in the central nervous system.