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Background: Triple‐negative breast cancer (TNBC) presents the most challenging subtype of all breast cancers because of its aggressive clinical phenotypes and absence of viable therapy targets. In order to identify effective molecular targets for treating patients with TNBC, we conducted an integration analysis of our recently published TNBC dataset of quantitative proteomics and RNA‐Sequencing, and found the abnormal upregulation of chromosome 9 open reading frame 142 (C9orf142) in TNBC. However, the functional roles of C9orf142 in TNBC are unclear. Methods: In vitro and in vivo functional experiments were performed to assess potential roles of C9orf142 in TNBC. Immunoblotting, real‐time quantitative polymerase chain reaction (RT‐qPCR), and immunofluorescent staining were used to investigate the expression levels of C9orf142 and its downstream molecules. The molecular mechanisms underlying C9orf142‐regulated mouse double minute 2 (MDM2)‐binding protein (MTBP) were determined by chromatin immunoprecipitation (ChIP) and dual‐luciferase reporter assays. Results: In TNBC tissues and metastatic lymph nodes, we observed that C9orf142 exhibited abnormal up‐regulation, and its elevated expression was indicative of unfavorable prognosis for TNBC patients. Both in vitro and in vivo functional experiments demonstrated that C9orf142 accelerated TNBC growth and metastasis. Further mechanism exploration revealed that C9orf142 transcriptionally activated MTBP, thereby regulating its downstream MDM2/p53/p21 signaling axis and the transition of cell cycle from G1 to S phase. Functional rescue experiment demonstrated that knockdown of MTBP attenuated C9orf142‐mediated tumour growth and metastasis. Furthermore, depletion of C9orf142 remarkably increased the responsiveness of TNBC cells to CDK4/6 inhibitor abemaciclib. Conclusions: Together, these findings unveil a previously unrecognized effect of C9orf142 in TNBC progression and responsiveness to CDK4/6 inhibitor, and emphasize C9orf142 as a promising intervention target for TNBC treatment. [ABSTRACT FROM AUTHOR]

Simple Summary: The MDM2 binding protein MTBP is implicated in various cellular functions and cancer-related processes, which vary depending on the cellular context and its localization within the cell. Moreover, the in vivo physiological function of MTBP remains unclear. To overcome embryonic lethality due to complete deletion of the Mtbp gene in mice, we created mice with an Mtbp hypomorphic allele (MtbpH) that expresses Mtbp protein at approximately 30% of the wild-type level. In a carcinogen-induced liver cancer model, MtbpH/− mice showed worse overall survival than wild-type mice. MEFs generated from the MtbpH/− mice displayed an increased nuclear localization of p-Erk1/2 protein and enhanced migratory potential. Thus, the newly generated MtbpH/− mice and MEFs can be used to study the in vivo physiological function of Mtbp and validate its diverse functions observed in human cells. MTBP is implicated in cell cycle progression, DNA replication, and cancer metastasis. However, the function of MTBP remains enigmatic and is dependent on cellular contexts and its cellular localization. To understand the in vivo physiological role of MTBP, it is important to generate Mtbp knockout mice. However, complete deletion of the Mtbp gene in mice results in early embryonic lethality, while its heterozygous deletion shows modest biological phenotypes, including enhanced cancer metastasis. To overcome this and better characterize the in vivo physiological function of MTBP, we, for the first time, generated mice that carry an Mtbp hypomorphic allele (MtbpH) in which Mtbp protein is expressed at approximately 30% of that in the wild-type allele. We treated wild-type, Mtbp+/−, and MtbpH/− mice with a liver carcinogen, diethylnitrosamine (DEN), and found that the MtbpH/− mice showed worse overall survival when compared to the wild-type mice. Consistent with previous reports using human liver cancer cells, mouse embryonic fibroblasts (MEFs) from the MtbpH/− mice showed an increase in the nuclear localization of p-Erk1/2 and migratory potential. Thus, MtbpH/− mice and cells from MtbpH/− mice are valuable to understand the in vivo physiological role of Mtbp and validate the diverse functions of MTBP that have been observed in human cells. [ABSTRACT FROM AUTHOR]

Abstract Background Triple‐negative breast cancer (TNBC) presents the most challenging subtype of all breast cancers because of its aggressive clinical phenotypes and absence of viable therapy targets. In order to identify effective molecular targets for treating patients with TNBC, we conducted an integration analysis of our recently published TNBC dataset of quantitative proteomics and RNA‐Sequencing, and found the abnormal upregulation of chromosome 9 open reading frame 142 (C9orf142) in TNBC. However, the functional roles of C9orf142 in TNBC are unclear. Methods In vitro and in vivo functional experiments were performed to assess potential roles of C9orf142 in TNBC. Immunoblotting, real‐time quantitative polymerase chain reaction (RT‐qPCR), and immunofluorescent staining were used to investigate the expression levels of C9orf142 and its downstream molecules. The molecular mechanisms underlying C9orf142‐regulated mouse double minute 2 (MDM2)‐binding protein (MTBP) were determined by chromatin immunoprecipitation (ChIP) and dual‐luciferase reporter assays. Results In TNBC tissues and metastatic lymph nodes, we observed that C9orf142 exhibited abnormal up‐regulation, and its elevated expression was indicative of unfavorable prognosis for TNBC patients. Both in vitro and in vivo functional experiments demonstrated that C9orf142 accelerated TNBC growth and metastasis. Further mechanism exploration revealed that C9orf142 transcriptionally activated MTBP, thereby regulating its downstream MDM2/p53/p21 signaling axis and the transition of cell cycle from G1 to S phase. Functional rescue experiment demonstrated that knockdown of MTBP attenuated C9orf142‐mediated tumour growth and metastasis. Furthermore, depletion of C9orf142 remarkably increased the responsiveness of TNBC cells to CDK4/6 inhibitor abemaciclib. Conclusions Together, these findings unveil a previously unrecognized effect of C9orf142 in TNBC progression and responsiveness to CDK4/6 inhibitor, and emphasize C9orf142 as a promising intervention target for TNBC treatment.

Simple Summary: Cancer is the second leading cause of death globally, accounting for approximately one in six deaths. Due to its critical role in cell growth/proliferation, one of the most well-known cancer-promoting proteins is MYC. Because cancer cells need MYC to grow, MYC is usually overactive in human cancers. However, despite decades of research, drugs targeting MYC continue to remain elusive. Notably, MYC requires other proteins to function properly. Our investigations into a protein named MTBP revealed that it associates with MYC and helps its pro-growth and cancer promoting function. Here, we discuss MTBP and review evidence showing MTBP is a critical partner for MYC, although the full extent of this partnership remains unresolved. We also discuss the role of MTBP in cancer, as MTBP levels are elevated in many human cancers and is often associated with reduced cancer patient survival. Additionally, we will discuss the inhibition of cancer cell growth and induction of cancer cell death by decreasing MTBP levels, indicating MTBP may represent a new cancer drug target and a way of indirectly targeting MYC. The oncogenic transcription factor c-MYC (MYC) is highly conserved across species and is frequently overexpressed or dysregulated in human cancers. MYC regulates a wide range of critical cellular and oncogenic activities including proliferation, metabolism, metastasis, apoptosis, and differentiation by transcriptionally activating or repressing the expression of a large number of genes. This activity of MYC is not carried out in isolation, instead relying on its association with a myriad of protein cofactors. We determined that MDM Two Binding Protein (MTBP) indirectly binds MYC and is a novel MYC transcriptional cofactor. MTBP promotes MYC-mediated transcriptional activity, proliferation, and cellular transformation by binding in a protein complex with MYC at MYC-bound promoters. This discovery provided critical context for data linking MTBP to aging as well as a rapidly expanding body of evidence demonstrating MTBP is overexpressed in many human malignancies, is often linked to poor patient outcomes, and is necessary for cancer cell survival. As such, MTBP represents a novel and potentially broad reaching oncologic drug target, particularly when MYC is dysregulated. Here we have reviewed the discovery of MTBP and the initial controversy with its function as well as its associations with proliferation, MYC, DNA replication, aging, and human cancer. [ABSTRACT FROM AUTHOR]

Simple Summary: Copying the chromosomal DNA completely and faithfully during the process of DNA replication is key to inheriting the genetic information in an unaltered state. To achieve this, replication initiation, which generates the complex molecular machines on the chromosomes that copy the DNA, must be precisely regulated. Despite our profound understanding of bacterial and viral DNA replication, our knowledge about the molecular processes and cellular regulation of replication initiation in eukaryotic cells was surprisingly scarce for a long time. Recently, fundamental progress has been made by studying purified replication initiation proteins structurally and biochemically. The MTBP protein (Mdm2-binding protein) was the last replication initiation factor to be identified in higher multicellular eukaryotes. MTBP is the counterpart of the Sld7 protein found in the simpler eukaryote budding yeast. MTBP is essential for replication initiation in cultured human cells and is emerging as a molecular relay for signals that control replication initiation at origins, such as initiation efficiency, placement and timing. We here discuss recent progress to unravel the molecular initiation processes and the role of MTBP. The initiation step of replication at replication origins determines when and where in the genome replication machines, replisomes, are generated. Tight control of replication initiation helps facilitate the two main tasks of genome replication, to duplicate the genome accurately and exactly once each cell division cycle. The regulation of replication initiation must ensure that initiation occurs during the S phase specifically, that no origin fires more than once per cell cycle, that enough origins fire to avoid non-replicated gaps, and that the right origins fire at the right time but only in favorable circumstances. Despite its importance for genetic homeostasis only the main molecular processes of eukaryotic replication initiation and its cellular regulation are understood. The MTBP protein (Mdm2-binding protein) is so far the last core replication initiation factor identified in metazoan cells. MTBP is the orthologue of yeast Sld7. It is essential for origin firing, the maturation of pre-replicative complexes (pre-RCs) into replisomes, and is emerging as a regulation focus targeted by kinases and by regulated degradation. We present recent insight into the structure and cellular function of the MTBP protein in light of recent structural and biochemical studies revealing critical molecular details of the eukaryotic origin firing reaction. How the roles of MTBP in replication and other cellular processes are mutually connected and are related to MTBP's contribution to tumorigenesis remains largely unclear. [ABSTRACT FROM AUTHOR]

Colon cancer is a malignant tumor that seriously affects human health. Recently, studies revealed that the expression of MTBP enhanced the proliferation and metastasis of many types of cancer cells. And the data also showed that MTBP has the potential to regulate the expression of ZEB2. However, it is unclear whether MTBP can affect the proliferation, migration and invasion of colon cancer cells by modulating the expression of ZEB2. In this study, we established the MTBP overexpression and knockdown colon cancer cells with the transfection. Next, CCK-8 and transwell assays were carried out to determine the changes of the proliferation and invasion of colon cancer cells, respectively. After that, we overexpressed the ZEB2 in these MTBP knockdown colon cancer cells. Finally, the invasion and migration of these cells were detected with the same methods. We revealed that overexpression of MTBP enhanced the proliferation and invasion of colon cancer cells. Moreover, suppression of MTBP repressed the proliferation, migration and invasion of colon cancer cells. Furthermore, MTBP promoted the expression of ZEB2. The overexpression of ZEB2 abolished the MTBP knockdown induced inhibition of the migration and invasion of colon cancer cells. These results implied that MTBP enhanced the proliferation, migration and invasion of colon cancer cells by activating the expression of ZEB2.

Treslin/Ticrr is required for the initiation of DNA replication and binds to MTBP (Mdm2 Binding Protein). Here, we show that in Xenopus egg extract, MTBP forms an elongated tetramer with Treslin containing two molecules of each protein. Immunodepletion and add-back experiments show that Treslin–MTBP is rate limiting for replication initiation. It is recruited onto chromatin before S phase starts and recruitment continues during S phase. We show that DDK activity both increases and strengthens the interaction of Treslin–MTBP with licensed chromatin. We also show that DDK activity cooperates with CDK activity to drive the interaction of Treslin–MTBP with TopBP1 which is a regulated crucial step in pre-initiation complex formation. These results suggest how DDK works together with CDKs to regulate Treslin–MTBP and plays a crucial in selecting which origins will undergo initiation.

Colon cancer is a malignant tumor that seriously affects human health. Recently, studies revealed that the expression of MTBP enhanced the proliferation and metastasis of many types of cancer cells. And the data also showed that MTBP has the potential to regulate the expression of ZEB2. However, it is unclear whether MTBP can affect the proliferation, migration and invasion of colon cancer cells by modulating the expression of ZEB2. In this study, we established the MTBP overexpression and knockdown colon cancer cells with the transfection. Next, CCK-8 and transwell assays were carried out to determine the changes of the proliferation and invasion of colon cancer cells, respectively. After that, we overexpressed the ZEB2 in these MTBP knockdown colon cancer cells. Finally, the invasion and migration of these cells were detected with the same methods. We revealed that overexpression of MTBP enhanced the proliferation and invasion of colon cancer cells. Moreover, suppression of MTBP repressed the proliferation, migration and invasion of colon cancer cells. Furthermore, MTBP promoted the expression of ZEB2. The overexpression of ZEB2 abolished the MTBP knockdown induced inhibition of the migration and invasion of colon cancer cells. These results implied that MTBP enhanced the proliferation, migration and invasion of colon cancer cells by activating the expression of ZEB2. [ABSTRACT FROM AUTHOR]

• Circ_0000119 is an exonic circular RNA deriving from protein-coding locus MAN1A2. • Circ_0000119 promotes gastric cancer cells proliferation and migration in vitro and in vivo. • miR-502-5p is sponged by circ_0000119 and thus MTBP is upregulated, favoring gastric cancer progression. Dysregulated circular RNAs (circRNAs) are significantly related with tumor initiation and progression. However, biological activity and potential molecular mechanism of circRNAs in gastric cancer (GC) deserve further exploration. We carried out total RNA sequencing and acquired the expression profiles of circRNAs. Quantitative real-time PCR as well as RNA in situ hybridization helped to validate circ_0000119 dysregulation. Various in vitro experiments were utilized to investigate the biological activities of circ_0000119 in GC, and the clinical relation of circ_0000119 in vivo was identified through nude mouse xenograft models. Finally, the molecular mechanism of circ_0000119 was clarified via luciferase assays, western blot, and rescue experiments. Compared with adjacent normal tissues, the study found an increase in the expression of circ_0000119 as well as its host linear gene MAN1A2 in GC tissues. Circ_0000119 overexpression promoted proliferation and migration of GC cells in vitro and in vivo, whereas circ_0000119 suppression had the opposite effect. Mechanistically, circ_0000119 sponged miR-502-5p which played an inhibitory role in tumors. Furthermore, we found that miR-502-5p alleviated GC progression through targeting MTBP and downregulating its expression at mRNA and protein levels. In conclusion, our findings reveal a new regulatory mechanism for circ_0000119, which sponges the miR-502-5p, suppresses MTBP expression, and finally promotes GC progression. [ABSTRACT FROM AUTHOR]

The oncogenic transcription factor c-MYC (MYC) is highly conserved across species and is frequently overexpressed or dysregulated in human cancers. MYC regulates a wide range of critical cellular and oncogenic activities including proliferation, metabolism, metastasis, apoptosis, and differentiation by transcriptionally activating or repressing the expression of a large number of genes. This activity of MYC is not carried out in isolation, instead relying on its association with a myriad of protein cofactors. We determined that MDM Two Binding Protein (MTBP) indirectly binds MYC and is a novel MYC transcriptional cofactor. MTBP promotes MYC-mediated transcriptional activity, proliferation, and cellular transformation by binding in a protein complex with MYC at MYC-bound promoters. This discovery provided critical context for data linking MTBP to aging as well as a rapidly expanding body of evidence demonstrating MTBP is overexpressed in many human malignancies, is often linked to poor patient outcomes, and is necessary for cancer cell survival. As such, MTBP represents a novel and potentially broad reaching oncologic drug target, particularly when MYC is dysregulated. Here we have reviewed the discovery of MTBP and the initial controversy with its function as well as its associations with proliferation, MYC, DNA replication, aging, and human cancer.

The initiation step of replication at replication origins determines when and where in the genome replication machines, replisomes, are generated. Tight control of replication initiation helps facilitate the two main tasks of genome replication, to duplicate the genome accurately and exactly once each cell division cycle. The regulation of replication initiation must ensure that initiation occurs during the S phase specifically, that no origin fires more than once per cell cycle, that enough origins fire to avoid non-replicated gaps, and that the right origins fire at the right time but only in favorable circumstances. Despite its importance for genetic homeostasis only the main molecular processes of eukaryotic replication initiation and its cellular regulation are understood. The MTBP protein (Mdm2-binding protein) is so far the last core replication initiation factor identified in metazoan cells. MTBP is the orthologue of yeast Sld7. It is essential for origin firing, the maturation of pre-replicative complexes (pre-RCs) into replisomes, and is emerging as a regulation focus targeted by kinases and by regulated degradation. We present recent insight into the structure and cellular function of the MTBP protein in light of recent structural and biochemical studies revealing critical molecular details of the eukaryotic origin firing reaction. How the roles of MTBP in replication and other cellular processes are mutually connected and are related to MTBP’s contribution to tumorigenesis remains largely unclear.

Summary: The processes that control where higher eukaryotic cells initiate DNA replication throughout the genome are not understood clearly. In metazoans, the Treslin-MTBP complex mediates critical final steps in formation of the activated replicative helicase prior to initiation of replication. Here, we map the genome-wide distribution of the MTBP subunit of this complex in human cells. Our results indicate that MTBP binds to at least 30,000 sites in the genome. A majority of these sites reside in regions of open chromatin that contain transcriptional-regulatory elements (e.g., promoters, enhancers, and super-enhancers), which are known to be preferred areas for initiation of replication. Furthermore, many binding sites encompass two genomic features: a nucleosome-free DNA sequence (e.g., G-quadruplex DNA or AP-1 motif) and a nucleosome bearing histone marks characteristic of open chromatin, such as H3K4me2. Taken together, these findings indicate that Treslin-MTBP associates coordinately with multiple genomic signals to promote initiation of replication.

Abstract Background Autoinflammatory diseases in dogs are characterized by complex disease processes with varying clinical signs. In Shar-Pei, signs of inflammation including fever and arthritis are known to be related with a breed-specific predisposition for Shar-Pei Autoinflammatory Disease (SPAID). Results Clinical and histopathological examinations of two severely SPAID-affected Shar-Pei revealed signs of inflammation including fever, arthritis, and perivascular and diffuse dermatitis in both dogs. A multifocal accumulation of amyloid in different organs was found in one SPAID-affected case. Whole genome sequencing resulted in 37 variants, which were homozygous mutant private mutations in SPAID-affected Shar-Pei. Nine SNVs with predicted damaging effects and three INDELs were further investigated in 102 Shar-Pei affected with SPAID, 62 unaffected Shar-Pei and 162 controls from 11 different dog breeds. The results showed the missense variant MTBP:g.19383758G > A in MTBP to be highly associated with SPAID in Shar-Pei. In the region of this gene a large ROH (runs of homozygosity) region could be detected exclusively in the two investigated SPAID-affected Shar-Pei compared to control dog breeds. No further SPAID-associated variant with predicted high or moderate effects could be found in genes identified in ROH regions. This MTBP variant was predicted to affect the MDN2-binding protein domain and consequently promote proinflammatory reactions. In the investigated group of Shar-Pei older than six years all dogs with the mutant genotype A/A were SPAID-affected whereas SPAID-unaffected dogs harbored the homozygous wildtype (G/G). Shar-Pei with a heterozygous genotype (G/A) were shown to have a 2.13-fold higher risk for disease development, which gave evidence for an incomplete dominant mode of inheritance. Conclusions The results of this study give strong evidence for a variant in MTBP related with proinflammatory processes via MTBP-MDM2 pathway. Thus, these results enable a reliable detection of SPAID in Shar-Pei dogs.

Background: It is clearly necessary to discover prognostic biomarkers to identify stage I patients at risk of recurrence and give them timely postoperative treatment. Materials and methods: Data of stage I lung adenocarcinoma were retrieved from four gene series in Gene Expression Omnibus (GEO) database (GSE50081, GSE30219, GSE37745, and GSE13213). Partek Genomics Suite software was used to identify survival-related genes for finding candidate indicators for early-stage patients at risk of recurrence. Differential expression of MTBP (MDM2 binding protein) in early-stage lung adenocarcinoma tissues was determined by immunohistochemical staining. The effects of MTBP interference expression and overexpression on viability, migration, and invasion capacity of lung cells were evaluated using Cell Counting Kit-8, wound healing, and Transwell assays. The tumor growth and lung metastasis in vivo were observed in chick embryo chorioallantoic membrane model. Human Exon 2.0 ST Array was used to analyze downstream regulation genes of MTBP in lung cancer cells. Involvement of ZEB2 and epithelial–mesenchymal transition (EMT) markers was investigated by Western blot. Results: By mining GEO database, we identified MTBP as a poor prognostic indicator of stage I lung adenocarcinomas. In addition, increased expression of MTBP was also associated with poor survival in our early-stage lung adenocarcinoma cohort. Further experiment suggested that knockdown of MTBP suppressed the migration and invasion of A549 and H1975 cells in vitro and in vivo, whereas overexpression of MTBP in HCC827 and PC9 cells promoted the migration and invasion in vitro and in vivo. Furthermore, ZEB2 upregulation directly activated EMT to mediate the downstream effects of MTBP involved in lung cancer cells metastasis. Conclusion: MTBP is an independent indicator for poor prognosis in stage I lung adenocar-cinomas and might promote the aggressive phenotype of non-small-cell lung cancer by inducing the EMT process through upregulating ZEB2 expression. [ABSTRACT FROM AUTHOR]

Background: Autoinflammatory diseases in dogs are characterized by complex disease processes with varying clinical signs. In Shar-Pei, signs of inflammation including fever and arthritis are known to be related with a breedspecific predisposition for Shar-Pei Autoinflammatory Disease (SPAID). Results: Clinical and histopathological examinations of two severely SPAID-affected Shar-Pei revealed signs of inflammation including fever, arthritis, and perivascular and diffuse dermatitis in both dogs. A multifocal accumulation of amyloid in different organs was found in one SPAID-affected case. Whole genome sequencing resulted in 37 variants, which were homozygous mutant private mutations in SPAID-affected Shar-Pei. Nine SNVs with predicted damaging effects and three INDELs were further investigated in 102 Shar-Pei affected with SPAID, 62 unaffected Shar-Pei and 162 controls from 11 different dog breeds. The results showed the missense variant MTBP:g.19383758G > A in MTBP to be highly associated with SPAID in Shar-Pei. In the region of this gene a large ROH (runs of homozygosity) region could be detected exclusively in the two investigated SPAID-affected Shar-Pei compared to control dog breeds. No further SPAID-associated variant with predicted high or moderate effects could be found in genes identified in ROH regions. This MTBP variant was predicted to affect the MDN2-binding protein domain and consequently promote proinflammatory reactions. In the investigated group of Shar-Pei older than six years all dogs with the mutant genotype A/A were SPAID-affected whereas SPAID-unaffected dogs harbored the homozygous wildtype (G/G). Shar-Pei with a heterozygous genotype (G/A) were shown to have a 2.13-fold higher risk for disease development, which gave evidence for an incomplete dominant mode of inheritance. Conclusions: The results of this study give strong evidence for a variant in MTBP related with proinflammatory processes via MTBP-MDM2 pathway. Thus, these results enable a reliable detection of SPAID in Shar-Pei dogs. [ABSTRACT FROM AUTHOR]

Background: Emerging evidence suggests that MTBP plays a role in cancer development and possibly progression, but its influence on hepatocellular carcinoma (HCC) remains unclear.Methods: We used real-time PCR and Western blotting to investigate MTBP expression in four HCC cell lines, 120 pairs of tumor and corresponding paracarcinomatous tissues from HCC patients. Immunohistochemistry was performed to examine MTBP expression in HCC and corresponding paracarcinomatous tissues from 120 patients. E-cadherin was only examined in HCC tissues of patients mentioned above. Statistical analyses were applied to evaluate the prognostic value and associations of MTBP expression with clinical parameters. Furthermore, the MTBP gene was overexpressed in HepG2 cell and silenced by siRNA in Hu7 cell, and cell migration and invasion were detected in vitro and in vivo. Moreover, the molecular mechanism of E-cadherin regulation by MTBP was explored.Results: In this study, we first showed that MTBP protein expression is positively correlated with distant metastasis and poor prognosis in HCC patients. We also found that MTBP expression was increased in metastatic cell lines when compared with nonmetastatic cell lines. Consistent with these findings, enhanced expression of MTBP promoted HCC cell invasiveness and metastasis both in vitro and in vivo, whereas the knockdown of MTBP with small interfering RNA resulted in reduced HCC migration and invasion. Ectopic expression of MTBP in HCC cells induced epithelial-to-mesenchymal transition, whereas the silencing of MTBP had the opposite effect. Furthermore, our results show that MTBP and E-cadherin protein expression are inversely correlated in primary HCC tissues. Moreover, our findings indicate that MTBP overexpression decreases E-cadherin expression through the modulation of Mdm2 ubiquitination degradation.Conclusions: Our data show that MTBP aggravates the invasion and metastasis of HCC by promoting the MDM2-mediated degradation of E-cadherin. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide with increasing incidence. Despite curative surgical resection and advanced chemotherapy, its survival rate remains low. The presence of microvascular invasion and occult metastasis is one of the major causes for this poor outcome. MDM2 Binding Protein (MTBP) has been implicated in the suppression of cell migration and cancer metastasis. However, clinical significance of MTBP, particularly in human cancer, is poorly understood. Specifically, clinical relevance of MTBP in human HCC has never been investigated. Here we demonstrated that expression of MTBP was significantly reduced in human HCC tissues compared to adjacent non-tumor tissues. MTBP expression was negatively correlated with capsular/vascular invasion and lymph node metastasis. Overexpression of MTBP resulted in the suppression of the migratory and metastatic potential of HCC cells, while its downregulation increased the migration. Consistent with the previous report, MTBP endogenously bound to alpha-actinin 4 (ACTN4) and suppressed ACTN4-mediated cell migration in multiple HCC cell lines. However, MTBP also inhibited migratory potential of PLC/PRF/5 HCC cells whose migration was not altered by manipulation of ACTN4 expression. These results suggest that mechanisms behind MTBP-mediated migration suppression may not be limited to the pathway involving ACTN4 in certain cellular contexts. Additionally, as a potential mechanism for reduced MTBP expression in tumors, we found that MTBP expression was increased following the treatment with histone deacetylase inhibitors (HDIs). Our study, for the first time, provides clinical relevance of MTBP in the suppression of HCC metastasis. [ABSTRACT FROM AUTHOR]

Metastasis is the process of primary tumor cells breaking away and colonizing distant secondary sites. In order for a tumor cell growing in one microenvironment to travel to, and flourish in, a secondary environment, it must survive a series of events termed the metastatic cascade. Before departing the primary tumor, cells acquire genetic and epigenetic changes that endow them with properties not usually associated with related normal differentiated cells. Those cells also induce a subset of bone marrow-derived stem cells to mobilize and establish pre-metastatic niches []. Many tumor cells undergo epithelial-to-mesenchymal transition (EMT), where they transiently acquire morphologic changes, reduced requirements for cell-cell contact and become more invasive []. Invasive tumor cells eventually enter the circulatory (hematogenous) or lymphatic systems or travel across body cavities. In transit, tumor cells must resist anoikis, survive sheer forces and evade detection by the immune system. For blood-borne metastases, surviving cells then arrest or adhere to endothelial linings before either proliferating or extravasating. Eventually, tumor cells complete the process by proliferating to form a macroscopic mass []. Up to 90 % of all cancer related morbidity and mortality can be attributed to metastasis. Surgery manages to ablate most primary tumors, especially when combined with chemotherapy and radiation. But if cells have disseminated, survival rates drop precipitously. While multiple parameters of the primary tumor are predictive of local or distant relapse, biopsies remain an imperfect science. The introduction of molecular and other biomarkers [, ] continue to improve the accuracy of prognosis. However, the invasive procedure introduces new complications for the patient. Likewise, the heterogeneity of any tumor population [, , ] means that sampling error (i.e., since it is impractical to examine the entire tumor) necessitates further improvements. In the case of breast cancer, for example, women diagnosed with stage I diseases (i.e., no evidence of invasion through a basement membrane) still have a ~30 % likelihood of developing distant metastases []. Many physicians and patients opt for additional chemotherapy in order to 'mop up' cells that have disseminated and have the potential to grow into macroscopic metastases. This means that ~ 70 % of patients receive unnecessary therapy, which has undesirable side effects. Therefore, improving prognostic capability is highly desirable. Recent advances allow profiling of primary tumor DNA sequences and gene expression patterns to define a so-called metastatic signature [-], which can be predictive of patient outcome. However, the genetic changes that a tumor cell must undergo to survive the initial events of the metastatic cascade and colonize a second location belie a plasticity that may not be adequately captured in a sampling of heterogeneous tumors. In order to tailor or personalize patient treatments, a more accurate assessment of the genetic profile in the metastases is needed. Biopsy of each individual metastasis is not practical, safe, nor particularly cost-effective. In recent years, there has been a resurrection of the notion to do a 'liquid biopsy,' which essentially involves sampling of circulating tumor cells (CTC) and/or cell free nucleic acids (cfDNA, including microRNA (miRNA)) present in blood and lymph [-]. The rationale for liquid biopsy is that tumors shed cells and/or genetic fragments into the circulation, theoretically making the blood representative of not only the primary tumor but also distant metastases. Logically, one would predict that the proportion of CTC and/or cfDNA would be proportionate to the likelihood of developing metastases []. While a linear relationship does not exist, the information within CTC or cfDNA is beginning to show great promise for enabling a global snapshot of the disease. However, the CTC and cfDNA are present at extremely low levels. Nonetheless, newer technologies capture enough material to enrich and sequence the patient's DNA or quantification of some biomarkers. Among the biomarkers showing great promise are metastasis suppressors which, by definition, block a tumor cell's ability to complete the metastatic process without prohibiting primary tumor growth []. Since the discovery of the first metastasis suppressor, Nm23, more than 30 have been functionally characterized. They function at various stages of the metastatic cascade, but their mechanisms of action, for the most part, remain ill-defined. Deciphering the molecular interactions of functional metastasis suppressors may provide insights for targeted therapies when these regulators cease to function and result in metastatic disease. In this brief review, we summarize what is known about the various metastasis suppressors and their functions at individual steps of the metastatic cascade (Table 1). Some of the subdivisions are rather arbitrary in nature, since many metastasis suppressors affect more than one step in the metastatic cascade. Nonetheless what emerges is a realization that metastasis suppressors are intimately associated with the microenvironments in which cancer cells find themselves []. [ABSTRACT FROM AUTHOR]

It has been proposed that ATR kinase senses the completion of DNA replication to initiate the S/G2 transition. In contrast to this model, we show here that the TRESLIN-MTBP complex prevents a premature entry into G2 from early S-phase independently of ATR/CHK1 kinases. TRESLIN-MTBP acts transiently at pre-replication complexes (preRCs) to initiate origin firing and is released after the subsequent recruitment of CDC45. This dynamic behavior of TRESLIN-MTBP implements a monitoring system that checks the activation of replication forks and senses the rate of origin firing to prevent the entry into G2. This system detects the decline in the number of origins of replication that naturally occurs in very late S, which is the signature that cells use to determine the completion of DNA replication and permit the S/G2 transition. Our work introduces TRESLIN-MTBP as a key player in cell-cycle control independent of canonical checkpoints., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

MDM2 binding protein (MTBP) is a protein that interacts with oncoprotein murine double minute (MDM2), a major inhibitor of the tumor suppressor p53. Overexpression of MTBP leads to p53-independent cell proliferation arrest, which is in turn blocked by simultaneous overexpression of MDM2. Importantly, reduced expression of MTBP in mice increases tumor metastasis and enhances migratory potential of mouse embryonic fibroblasts regardless of the presence of p53. Clinically, loss of MTBP expression in head and neck squamous cell carcinoma is associated with reduced patient survival, and is shown to serve as an independent prognostic factor when p53 is mutated in tumors. These results indicate the involvement of MTBP in suppressing tumor progression. Our recent findings demonstrate that overexpression of MTBP in human osteosarcoma cells lacking wild-type p53 inhibits metastasis, but not primary tumor growth, when cells are transplanted in femurs of immunocompromised mice. These data indicate that MTBP functions as a metastasis suppressor independent of p53 status. Furthermore, overexpression of MTBP suppresses cell migration and filopodia formation, in part, by inhibiting function of an actin crosslinking protein α-actinin-4. Thus, increasing evidence indicates the significance of MTBP in tumor progression. We summarize published results related to MTBP function and discuss caveats and future directions in this review article. [ABSTRACT FROM AUTHOR]

BACKGROUND: Recent genetic studies have implicated p53 mutation as a significant risk factor for therapeutic failure in squamous cell carcinoma of the head and neck (SCCHN). However, in a recent meta-analysis in the literature of p53 from major anatomical subsites (larynx, oral cavity, oropharynx/hypopharynx), associations between patient survival and p53 status were ambiguous. METHODS: The authors examined a cohort of SCCHNs using a previously developed biomarker combination that likely predicts p53 status based on p53/MDM2 expression levels determined by immunohistochemistry (IHC). In addition, the authors generated and validated an antibody to MTBP (an MDM2 binding protein that alters p53/MDM2 homeostasis and may contribute to metastatic suppression) and have incorporated data for MTBP expression into the current analyses. RESULTS: Analysis of expression data for p53 and MDM2 in 198 SCCHN patient samples revealed that the biomarker combination p53 + ve/MDM2-low (likely indicative of p53 mutation) was significantly associated with reduced overall survival (log-rank P = .035) and was an independent prognostic factor (P = .013; HR, 1.705; 95% CI, 1.12-2.60); thus, these data were compatible with earlier genetic analyses. By using IHC for p53 and MDM2 to dichotomize patients, the authors found that loss of MTBP expression was significantly associated with reduced survival (log-rank P = .004) and was an independent prognostic factor (P = .004; HR, 2.78; 95% CI, 1.39-5.54) in p53 + ve/MDM2-low patients. CONCLUSIONS: These results represent the first examination of MTBP expression in human tissues and provide evidence for a p53 status-dependent role for MTBP in suppressing disease progression in SCCHN patients as well as confirming a role for p53 pathway function in delaying disease progression. [ABSTRACT FROM AUTHOR]

Murine double minute 2 (MDM2) binding protein (MTBP) has been implicated in tumor cell proliferation, but the underlying mechanisms remain unclear. The results of MTBP expression analysis during cell cycle progression demonstrated that MTBP protein was rapidly degraded during mitosis. Immunofluorescence studies revealed that a portion of MTBP was localized at the kinetochores during prometaphase. MTBP overexpression delayed mitotic progression from nuclear envelope breakdown (NEB) to anaphase onset and induced abnormal chromosome segregation such as lagging chromosomes, chromosome bridges, and multipolar chromosome segregation. Conversely, MTBP downmodulation caused an abbreviated metaphase and insufficient mitotic arrest, resulting in abnormal chromosome segregation, aneuploidy, decreased cell proliferation, senescence, and cell death, similar to that of Mad2 (mitotic arrest-deficient 2) downmodulation. Furthermore, MTBP downmodulation inhibited the accumulation of Mad1 and Mad2, but not BubR1 (budding uninhibited by benzimidazoles related 1), on the kinetochores, whereas MTBP overexpression inhibited the release of Mad2 from the metaphase kinetochores. These results may imply that MTBP has an important role in recruiting and/or retaining the Mad1/Mad2 complex at the kinetochores during prometaphase, but its degradation is required for silencing the mitotic checkpoint. Together, this study indicates that MTBP has a crucial role in proper mitotic progression and faithful chromosome segregation, providing new insights into regulation of the mitotic checkpoint. [ABSTRACT FROM AUTHOR]

Mdm2 binding protein (MTBP) has been implicated in cell-cycle arrest and the Mdm2/p53 tumor suppressor pathway through its interaction with Mdm2. To determine the function of MTBP in tumorigenesis and its potential role in the Mdm2/p53 pathway, we crossed Mtbp-deficient mice to Eμ-myc transgenic mice, in which overexpression of the oncogene c-Myc induces B-cell lymphomas primarily through inactivation of the Mdm2/p53 pathway. We report that Myc-induced B-cell lymphoma development in Mtbp heterozygous mice was profoundly delayed. Surprisingly, reduced levels of Mtbp did not lead to an increase in B-cell apoptosis or affect Mdm2. Instead, an Mtbp deficiency inhibited Myc-induced proliferation and the upregulation of Myc target genes necessary for cell growth. Consistent with a role in proliferation, Mtbp expression was induced by Myc and other factors that promote cell-cycle progression and was elevated in lymphomas from humans and mice. Therefore, Mtbp functioned independent of Mdm2 and was a limiting factor for the proliferative and transforming functions of Myc. Thus, Mtbp is a previously unrecognized regulator of Myc-induced tumorigenesis. [ABSTRACT FROM AUTHOR]

Treslin/Ticrr is required for the initiation of DNA replication and binds to MTBP (Mdm2 Binding Protein). Here, we show that in Xenopus egg extract, MTBP forms an elongated tetramer with Treslin containing two molecules of each protein. Immunodepletion and add-back experiments show that Treslin-MTBP is rate limiting for replication initiation. It is recruited onto chromatin before S phase starts and recruitment continues during S phase. We show that DDK activity both increases and strengthens the interaction of Treslin-MTBP with licensed chromatin. We also show that DDK activity cooperates with CDK activity to drive the interaction of Treslin-MTBP with TopBP1 which is a regulated crucial step in pre-initiation complex formation. These results suggest how DDK works together with CDKs to regulate Treslin-MTBP and plays a crucial in selecting which origins will undergo initiation.

The processes that control where higher eukaryotic cells initiate DNA replication throughout the genome are not understood clearly. In metazoans, the Treslin-MTBP complex mediates critical final steps in formation of the activated replicative helicase prior to initiation of replication. Here, we map the genome-wide distribution of the MTBP subunit of this complex in human cells. Our results indicate that MTBP binds to at least 30,000 sites in the genome. A majority of these sites reside in regions of open chromatin that contain transcriptional-regulatory elements (e.g., promoters, enhancers, and super-enhancers), which are known to be preferred areas for initiation of replication. Furthermore, many binding sites encompass two genomic features: a nucleosome-free DNA sequence (e.g., G-quadruplex DNA or AP-1 motif) and a nucleosome bearing histone marks characteristic of open chromatin, such as H3K4me2. Taken together, these findings indicate that Treslin-MTBP associates coordinately with multiple genomic signals to promote initiation of replication., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background : Glioblastoma (GBM) is highly proliferative and resistant to radio-chemotherapy. Loss of tumor suppressor gene TP53 function frequently occurs at protein level in GBMs. This inhibition is often mediated by other components within the p53 signaling axis, including MDM2, whose binding protein (MTBP) plays an important role in the regulation of MDM2 and p53 activity. We investigated the role of MTBP in the biology of TP53-wildtype (TP53wt) GBMs. Methods : MTBP expression was examined in TCGA and REMBRANDT datasets. MTBP was silenced or overexpressed in TP53wt GBM cells and glioma stem cells (GSCs). The effects on cell viability, apoptosis, and clonogenicity were assessed. The transcriptional regulation of MTBP was investigated. Results : Upregulation of MTBP was correlated with the Classical molecular subtype, and it predicted poor survival. In TP53wt GBM cells, the protein levels of MTBP were positively associated with those of MDM2 but negatively correlated with those of p53. MTBP knockdown promoted apoptosis and inhibited clonogenicity, while overexpression of this protein enhanced tumorigenicity in vitro and in vivo . The pro-survival effect of MTBP depended on the activity of MDM2 and p53. MTBP was transcriptionally regulated by c-myc, thereby forming a positive regulatory loop. Finally, MTBP silencing increased the sensitivity of TP53wt GSCs to radiation and TMZ treatment in vitro and in vivo . Conclusion : MTBP regulates the cell survival and treatment sensitivity of TP53wt GBMs through MDM2-dependent post-translational modification of p53. MTBP-targeting treatments are potentially useful in increasing patients' survival., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Objective: To investigate the role of MTBP in regulating the migration and invasion of human prostate cancer cells., Methods: The baseline expressions of MTBP in 3 different human prostate cancer cells lines (22RV1, DU145 and Lncap) were detected using Western blotting. The cells were transfected with a small interfering RNA (siRNA) for MTBP knockdown or MTBP plasmid for MTBP overexpression, and 48 h later, the cells were examined for MTBP expression with Western blotting; the changes in the migration abilities of the cells were evaluated using wound healing assay and Transwell assay, and the cell invasiveness was assessed using Matrigel Transwell assay. The expression of E-cadherin protein, a marker of epithelial mesenchymal transition (EMT), was detected using Western blotting., Results: MTBP expression was the highest in DU145 cells followed by Lncap cells, and was the lowest in 22RV1 cells, indicating a positive correlation of MTBP expression with the level of malignancy of human prostate cancer cells. Transfection of the cells with siRNA or MTBP plasmids efficiently lowered or enhanced the expressions of MTBP in human prostate cancer cells. Wound healing assay showed that inhibition of MTBP expression decreased the migration ability of the prostate cancer cells, and MTBP overexpression significantly promoted the migration of the cells ( P < 0.01). Transwell assay showed that MTBP knockdown significantly lowered the migration and invasion ability of the cells, while MTBP overexpression markedly increased the number of migrating and invading cells ( P < 0.01); Western blotting results showed that MTBP knockdown increased the expression of E-cadherin protein, and MTBP overexpression decreased E-cadherin expression in the prostate cancer cells., Conclusions: MTBP overexpression promotes the migration and invasion of human prostate cancer cells possibly relation to the induction of EMT.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and the prognosis of HCC patients, especially those with metastasis, remains extremely poor. This is partly due to unclear molecular mechanisms underlying HCC metastasis. Our previous study indicates that MDM2 Binding Protein (MTBP) suppresses migration and metastasis of HCC cells. However, signaling pathways regulated by MTBP remain unknown. To identify metastasis-associated signaling pathways governed by MTBP, we have performed unbiased luciferase reporter-based signal array analyses and found that MTBP suppresses the activity of the ETS-domain transcription factor Elk-1, a downstream target of Erk1/2 MAP kinases. MTBP also inhibits phosphorylation of Elk-1 and decreases mRNA expression of Elk-1 target genes. Reduced Elk-1 activity is caused by inhibited nuclear translocation of phosphorylated Erk1/2 (p-Erk) by MTBP and subsequent inhibition of Elk-1 phosphorylation. We also reveal that MTBP inhibits the interaction of p-Erk with importin-7/RanBP7 (IPO7), an importin family member which shuttles p-Erk into the nucleus, by binding to IPO7. Moreover, high levels of MTBP in human HCC tissues are correlated with cytoplasmic localization of p-Erk1/2. Our study suggests that MTBP suppresses metastasis, at least partially, by down-modulating the Erk1/2-Elk-1 signaling pathway, thus identifying a novel regulatory mechanism of HCC metastasis by regulating the subcellular localization of p-Erk., Competing Interests: CONFLICTS OF INTEREST No potential conflicts of interest were disclosed.

Alterations of specific genes can modulate aging. Myc, a transcription factor that regulates the expression of many genes involved in critical cellular functions was shown to have a role in controlling longevity. Decreased expression of Myc inhibited many of the deleterious effects of aging and increased lifespan in mice. Without altering Myc expression, reduced levels of Mtbp, a recently identified regulator of Myc, limit Myc transcriptional activity and proliferation, while increased levels promote Myc-mediated effects. To determine the contribution of Mtbp to the effects of Myc on aging, we studied a large cohort of Mtbp heterozygous mice and littermate matched wild-type controls. Mtbp haploinsufficiency significantly increased longevity and maximal survival in mice. Reduced levels of Mtbp did not alter locomotor activity, litter size, or body size, but Mtbp heterozygous mice did exhibit elevated markers of metabolism, particularly in the liver. Mtbp +/- mice. Therefore, the data suggest Mtbp is a regulator of longevity in mice that mimics some, but not all, of the properties of Myc in aging.Mtbp +/+ mice. Therefore, the data suggest Mtbp is a regulator of longevity in mice that mimics some, but not all, of the properties of Myc in aging., Competing Interests: The authors declare that they have no conflict of interest.