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.     

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.     

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.     

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。     

乳腺癌转移抑制因子BRMS1研究进展

癌转移是导致癌症患者死亡的主要原因, 因此, 研究癌转移的分子机制能为癌症的预后和治疗提供新的方法。癌转移抑制基因是一类只抑制癌细胞的转移而不影响肿瘤的发生与生长的基因。BRMS1是2000年在乳腺癌细胞中发现的癌转移抑制基因。它所编码的蛋白还可以抑制黑素瘤细胞和小鼠乳腺癌细胞的转移。BRMS1定位于核内, 与mSin3-HDAC复合体相互作用, 并且可以改变乳腺癌细胞的connexin表达特征, 从而恢复间隙连接介导的细胞间连接通讯。文章就BRMS1的研究进展做一综述, 对其相关的基因也给予简单的介绍, 并对它可能的作用机制进行了预测。     

肺癌转移抑制相关基因研究进展

肺癌是发病率和死亡率增长最快、对人类健康威胁最大的恶性肿瘤之一。侵袭转移是肺癌患者死亡的首要原因。研究表明,在肺癌中发挥转移抑制作用的相关基因主要有nm23、KAI1、TIMP、Cadherin以及MRP-1等。本文对近年来这些基因的研究进展及其对肺癌侵袭转移的抑制作用作一综述     

Identification of differential phosphorylation and sub-cellular localization of the metastasis suppressor,NDRG1

The metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), exhibits pleiotropic activity, inhibiting metastasis of various tumor-types, while being correlated with metastasis in others. Notably, NDRG1 phosphorylation and cleavage are associated with its function, although it is unclear if these modifications occur universally, or selectively, in different cancer cell-types and if it contributes to its pleiotropy. Considering the suggested DNA repair role of nuclear NDRG1, the effects of the above post-translational modifications on its nuclear localization was examined. Herein, the full-length (FL) and truncated (T) NDRG1 isoforms were detected using a C-terminus-directed antibody, while only the FL isoform was identified using an N-terminus-directed antibody. For the first time, we demonstrate that the expression of the NDRG1 FL and T forms occurs in all cancer cell-types examined, as does its phosphorylation (p-NDRG1) at Ser330 and Thr346. The FL isoform localized highly in the nucleus compared to the T isoform. Moreover, p-NDRG1 (Ser330) was also markedly localized in the nucleus, while p-NDRG1 (Thr346) was predominantly cytoplasmic in all cell-types. These results indicate the N-terminus region and phosphorylation at Ser330 could be crucial for NDRG1 nuclear localization and function. PTEN silencing indicated that p-NDRG1 (Thr346) could be regulated differentially in different tumor cell-types, indicating PTEN may be involved in the mechanism(s) underlying the pleiotropic activity of NDRG1. Finally, therapeutics of the di-2-pyridylketone thiosemicarbazone class increased nuclear NDRG1 isoforms (FL and T) detected by the C-terminus-directed antibody in HepG2 cells, while having no significant effect in PC3 cells, indicating differential activity depending on the cell-type.     

The tumor suppressor gene ARHI (DIRAS3) inhibits ovarian cancer cell migration through multiple mechanisms

ARHI is an imprinted tumor suppressor gene that is downregulated in > 60% of ovarian cancers, associated with decreased progression-free survival. ARHI encodes a 26 kDa GTPase with homology to Ras. Re-expression of ARHI inhibits ovarian cancer growth, initiates autophagy and induces tumor dormancy. Recent studies have demonstrated that ARHI also plays a particularly important role in ovarian cancer cell migration. Re-expression of ARHI decreases motility of IL-6- and EGF-stimulated SKOv3 and Hey ovarian cancer cells, inhibiting both chemotaxis and haptotaxis. ARHI inhibits cell migration by binding and sequestering STAT3 in the cytoplasm, and preventing STAT3 translocation to the nucleus and localization in focal adhesion complexes. Re-expression of ARHI inhibits FAK^Y397 phosphorylation, disrupts focal adhesions and blocks FAK-mediated RhoA signaling, resulting in decreased levels of GTP-RhoA. Re-expression of ARHI disrupts formation of actin stress fibers in a FAK- and RhoA-dependent manner. Recent studies indicate that re-expression of ARHI inhibits expression of β-1 integrin which may also contribute to inhibition of migration, adhesion and invasion.     

Cyclin-dependent kinase 1 (CDK1)-mediated phosphorylation of enhancer of zeste 2 (Ezh2) regulates its stability

The H3K27 histone methyltransferase, Ezh2 (enhancer of zeste 2), is a Polycomb group protein that plays important roles in many biological processes including cellular differentiation, stem cell biology, and cancer development. Up-regulation of Ezh2 is observed in various human cancers consistent with its role in cell proliferation. Thus, understanding the regulation of Ezh2 may reveal how it contributes to the cellular proliferation process. Here, we demonstrate that Ezh2 can be regulated by the cyclin-dependent kinase, CDK1, which phosphorylates Ezh2 at threonines 345 and 487. Consistent with the cell cycle phase during which CDK1 exhibits peak activity, Ezh2 phosphorylation is enriched in cells arrested in mitosis when compared with S-phase. Phosphorylation of Thr-345 and Thr-487 promotes Ezh2 ubiquitination and subsequent degradation by the proteasome. Furthermore, expression of T345A/T487A confers a proliferative disadvantage when compared with cells expressing wild-type Ezh2, which suggests that phosphorylation of Ezh2 is important for cell proliferation. Collectively, these results establish a novel function for CDK1-mediated Ezh2 phosphorylation and provide a mechanism by which Ezh2 protein levels can be regulated in cells.     

Myosin phosphatase targeting subunit 1 affects cell migration by regulating myosin phosphorylation and actin assembly

Myosin II plays important roles in many contractile-like cell functions, including cell migration, adhesion, and retraction. Myosin II is activated by regulatory light chain (RLC) phosphorylation whereas RLC dephosphorylation by myosin light chain phosphatase containing a myosin phosphatase targeting subunit (MYPT1) leads to myosin inactivation. HeLa cells contain MYPT1 in addition to a newly identified human variant 2 containing an internal deletion. RLC dephosphorylation, cell migration, and adhesion were inhibited when either or both MYPT1 isoforms were knocked down by RNA interference. RLC was highly phosphorylated (60%) when both isoforms were suppressed by siRNA treatment relative to control cells (10%) with serum-starvation and ROCK inhibition. Prominent stress fibers and focal adhesions were associated with the enhanced RLC phosphorylation. The reintroduction of MYPT1 or variant 2 in siRNA-treated cells decreased stress fibers and focal adhesions. MYPT1 knockdown also led to an increase of F-actin relative to G-actin in HeLa cells. The myosin inhibitor blebbistatin did not inhibit this effect, indicating MYPT1 likely affects actin assembly independent of RLC phosphorylation. Proper expression of MYPT1 or variant 2 is critical for RLC phosphorylation and actin assembly, thus maintaining normal cellular functions by simultaneously controlling cytoskeletal architecture and actomyosin activation.     

Inactivation of the p19(ARF) tumor suppressor affects intestinal epithelial cell proliferation and integrity

p19(ARF) is a tumor suppressor that is frequently deleted in human cancer. It lies at chromosome 9p21 and shares exons 2 and 3 with p16(ink4a), which is also inactivated by these cancer-associated deletions. The "canonical pathway" by which p19(ARF) is thought to suppress tumorigenesis through activation of the p53 tumor suppressor. In response to hyperproliferative signals, such as expression of oncogenes, p19(ARF) is induced and binds to the MDM2 ubiquitin ligase, sequestering it in the nucleolus to allow the accumulation of p53. However, p19(ARF) also has MDM2 and p53 independent functions. In human colon cancer, p19(ARF) is only rarely deleted, but it is more frequently silenced by DNA promoter methylation. Here we show that inactivation of p19(ARF) in mice increases the number of cycling cells in the crypts of the colonic epithelium. Moreover, inactivation of p19(ARF) exacerbated the ulceration of the colonic epithelium caused by dextran sodium sulfate (DSS). These effects were similar to those observed in mice lacking myeloid translocation gene-related-1 (Mtgr1), and mice lacking both of these genes showed an even greater sensitivity to DSS. Surprisingly, inactivation of p19(ARF) restored the loss of the secretory lineage in mice deficient in Mtgr1, suggesting an additional role for p19(ARF) in the small intestinal epithelium.     

SMURF1 Plays a role in EGF-induced breast cancer cell migration and invasion

Epidermal growth factor (EGF) is a well-known growth factor that induces cancer cell migration and invasion. Previous studies have shown that SMAD ubiquitination regulatory factor 1 (SMURF1), an E3 ubiquitin ligase, regulates cell motility by inducing RhoA degradation. Therefore, we examined the role of SMURF1 in EGF-induced cell migration and invasion using MDA-MB-231 cells, a human breast cancer cell line. EGF increased SMURF1 expression at both the mRNA and protein levels. All ErbB family members were expressed in MDA-MB-231 cells and receptor tyrosine kinase inhibitors specific for the EGF receptor (EGFR) or ErbB2 blocked the EGF-mediated induction of SMURF1 expression. Within the signaling pathways examined, ERK1/2 and protein kinase C activity were required for EGF-induced SMURF1 expression. The overexpression of constitutively active MEK1 increased the SMURF1 to levels similar to those induced by EGF. SMURF1 induction by EGF treatment or by the overexpression of MEK1 or SMURF1 resulted in enhanced cell migration and invasion, whereas SMURF1 knockdown suppressed EGF- or MEK1-induced cell migration and invasion. EGF treatment or SMURF1 overexpression decreased the endogenous RhoA protein levels. The overexpression of constitutively active RhoA prevented EGF- or SMURF1-induced cell migration and invasion. These results suggest that EGFinduced SMURF1 plays a role in breast cancer cell migration and invasion through the downregulation of RhoA.     

Inhibition of CDK-mediated Smad3 phosphorylation reduces the Pin1-Smad3 interaction and aggressiveness of triple negative breast cancer cells

Triple negative breast cancer (TNBC) is a highly aggressive breast cancer subtype that lacks effective targeted therapies. Although TNBC is not defined by specific therapeutic targets, a subset of patients have tumors that overexpress cyclins. High cyclin D/E expression catalyzes CDK4/2 activity. In turn, CDK4/2 can non-canonically phosphorylate Smad3, a key TGFβ signaling intermediate, and this phosphorylation has been associated with the shift from tumor-suppressive to oncogenic TGFβ pathway action in breast oncogenesis. Additionally, CDK-mediated Smad3 phosphorylation facilitates an interaction between Smad3 and Pin1, a cis-trans isomerase that is also overexpressed in aggressive breast cancers. Treatment with CYC065, a CDK2/9 inhibitor, decreased non-canonical Smad3 phosphorylation and inhibited the Pin1-Smad3 interaction. We hypothesized that the interaction of Pin1 and Smad3, facilitated by CDK-mediated Smad3 phosphorylation, promotes TNBC cell aggressiveness. Inhibition of the Pin1-Smad3 interaction in TNBC cell lines, through depletion of Pin1 or CYC065 treatment, resulted in decreased cell migration/invasion and impeded the EMT program. Inhibition of CDK-mediated phosphorylation of Smad3 by mutagenesis also decreased cell migration, underscoring the importance of non-canonical CDK2 phosphorylation of Smad3 to enable cell motility. Pin1 depletion restored Smad3 protein levels and tumor-suppressive activity, suggesting that the Pin1-Smad3 interaction has a negative impact on canonical Smad3 action. Collectively, the data show that the Pin1-Smad3 interaction, facilitated by CDK-mediated Smad3 phosphorylation, is associated with oncogenic TGFβ signaling and breast cancer progression. Inhibition of this interaction with CYC065 treatment may provide an important therapeutic option for TNBC patients.     

Poly (ADP-ribose) polymerase 1 (PARP1) inhibition promotes pulmonary metastasis of osteosarcoma by boosting ezrin phosphorylation

Due to the large proportion of BRCA deficiency and chromosomal instability in OS patients, poly (ADP-ribose) polymerase inhibitors (PARPi) could be an effective strategy for anti-OS therapy. In two orthotopic OS mouse models, we discovered that although PARPi had inhibitory effect on the growth of the orthotopic OS tumors regardless of BRCA deficiency, the treatment of PARPi essentially aggravated the pulmonary metastasis of OS in both models. A protein playing a crucial role in OS metastasis, ezrin, was identified as an interactive protein for PARP1. The phosphorylation of ezrin was significantly promoted during PARP inhibition. Besides the traditional function of phosphorylated ezrin at plasma membrane, we newly identified its nuclear speckle localization and its function with mRNA export. Ezrin knockdown or phosphorylation inhibition could partially rescue PARPi induced metastasis. Collectively, we unveiled a new mechanism for PARP-involved OS metastasis, which proposed a novel combinational therapy strategy using PARP and ezrin inhibitors for future OS treatment.