FKBP38 Regulates Self-Renewal and Survival of GBM Neurospheres.

Glioblastoma is the most common malignant primary brain tumor. The outcome is dismal, despite the multimodal therapeutic approach that includes surgical resection, followed by radiation and chemotherapy. The quest for novel therapeutic targets to treat glioblastoma is underway. FKBP38, a member of the immunophilin family of proteins, is a multidomain protein that plays an important role in the regulation of cellular functions, including apoptosis and autophagy. In this study, we tested the role of FKBP38 in glioblastoma tumor biology. Expression of FKBP38 was upregulated in the patient-derived primary glioblastoma neurospheres (GBMNS), compared to normal human astrocytes. Attenuation of FKBP38 expression decreased the viability of GBMNSs and increased the caspase 3/7 activity, indicating that FKBP38 is required for the survival of GBMNSs. Further, the depletion of FKBP38 significantly reduced the number of neurospheres that were formed, implying that FKBP38 regulates the self-renewal of GBMNSs. Additionally, the transient knockdown of FKBP38 increased the LC3-II/I ratio, suggesting the induction of autophagy with the depletion of FKBP38. Further investigation showed that the negative regulation of autophagy by FKBP38 in GBMNSs is mediated through the JNK/C-Jun-PTEN-AKT pathway. In vivo, FKBP38 depletion significantly extended the survival of tumor-bearing mice. Overall, our results suggest that targeting FKBP38 imparts an anti-glioblastoma effect by inducing apoptosis and autophagy and thus can be a potential therapeutic target for glioblastoma therapy.

Targeting protein arginine methyltransferase 5 sensitizes glioblastoma to trametinib.

Background: The prognosis of glioblastoma (GBM) remains dismal because therapeutic approaches have limited effectiveness. A new targeted treatment using MEK inhibitors, including trametinib, has been proposed to improve GBM therapy. Trametinib had a promising preclinical effect against several cancers, but its adaptive treatment resistance precluded its clinical translation in GBM. Previously, we have demonstrated that protein arginine methyltransferase 5 (PRMT5) is upregulated in GBM and its inhibition promotes apoptosis and senescence in differentiated and stem-like tumor cells, respectively. We tested whether inhibition of PRMT5 can enhance the efficacy of trametinib against GBM. Methods: Patient-derived primary GBM neurospheres (GBMNS) with transient PRMT5 knockdown were treated with trametinib and cell viability, proliferation, cell cycle progression, ELISA, and western blot were analyzed. In vivo, NSG mice were intracranially implanted with PRMT5-intact and -depleted GBMNS, treated with trametinib by daily oral gavage, and observed for tumor progression and mice survival rate. Results: PRMT5 depletion enhanced trametinib-induced cytotoxicity in GBMNS. PRMT5 knockdown significantly decreased trametinib-induced AKT and ERBB3 escape pathways. However, ERBB3 inhibition alone failed to block trametinib-induced AKT activity suggesting that the enhanced antitumor effect imparted by PRMT5 knockdown in trametinib-treated GBMNS resulted from AKT inhibition and not ERBB3 inhibition. In orthotopic murine xenograft models, PRMT5-depletion extended the survival of tumor-bearing mice, and combination with trametinib further increased survival. Conclusion: Combined PRMT5/MEK inhibition synergistically inhibited GBM in animal models and is a promising strategy for GBM therapy.

Pathological Features of Tumors of the Nervous System in Hereditary Cancer Predisposition Syndromes: A Review.

Hereditary cancer predisposition syndromes (HCS) become more recognizable as the knowledge about them expands, and genetic testing becomes more affordable. In this review, we discussed the known HCS that predispose to central and peripheral nervous system tumors. Different genetic phenomena were highlighted, and the important cellular biological alterations were summarized. Genetic mosaicism and germline mutations are features of HCS, and recently, they were described in normal population and as modifiers for the genetic landscape of sporadic tumors. Description of the tumors arising in these conditions was augmented by representative cases explaining the main pathological findings. Clinical spectrum of the syndromes and diagnostic criteria were tabled to outline their role in defining these disorders. Interestingly, precision medicine has found its way to help these groups of patients by offering targeted preventive measures. Understanding the signaling pathway alteration of mammalian target of rapamycin (mTOR) in tuberous sclerosis helped introducing mTOR inhibitors as a prophylactic treatment in these patients. More research to define the germline genetic alterations and resulting cellular signaling perturbations is needed for effective risk-reducing interventions beyond prophylactic surgeries.

Inhibiting protein phosphatase 2A increases the antitumor effect of protein arginine methyltransferase 5 inhibition in models of glioblastoma.

BACKGROUND: Despite multi-model therapy of maximal surgical resection, radiation, chemotherapy, and tumor-treating fields, the median survival of glioblastoma (GBM) patients is less than 15 months. Protein arginine methyltransferase 5 (PRMT5) catalyzes the symmetric dimethylation of arginine residues and is overexpressed in GBM. Inhibition of PRMT5 causes senescence in stem-like GBM tumor cells. LB100, a first-in-class small molecular inhibitor of protein phosphatase 2A (PP2A), can sensitize therapy-resistant tumor cells. Here, we tested the anti-GBM effect of concurrent PRMT5 and PP2A inhibition. METHODS: Patient-derived primary GBM neurospheres (GBMNS), transfected with PRMT5 target-specific siRNA, were treated with LB100 and subjected to in vitro assays including PP2A activity and western blot. The intracranial mouse xenograft model was used to test the in vivo antitumor efficacy of combination treatment. RESULTS: We found that PRMT5 depletion increased PP2A activity in GBMNS. LB100 treatment significantly reduced the viability of PRMT5-depleted GBMNS compared to PRMT5-intact GBMNS. LB100 enhanced G1 cell cycle arrest induced by PRMT5 depletion. Combination therapy also increased the expression of phospho-MLKL. Necrostatin-1 rescued PRMT5-depleted cells from the cytotoxic effects of LB100, indicating that necroptosis caused the enhanced cytotoxicity of combination therapy. In the in vivo mouse tumor xenograft model, LB100 treatment combined with transient depletion of PRMT5 significantly decreased tumor size and prolonged survival, while LB100 treatment alone had no survival benefit. CONCLUSION: Overall, combined PRMT5 and PP2A inhibition had significantly greater antitumor effects than PRMT5 inhibition alone.

Mutagenic Analysis of the C-Terminal Extension of Lsm1.

The Sm-like proteins (also known as Lsm proteins) are ubiquitous in nature and exist as hexa or heptameric RNA binding complexes. They are characterized by the presence of the Sm-domain. The Lsm1 through Lsm7 proteins are highly conserved in eukaryotes and they form a hetero-octameric complex together with the protein Pat1. The Lsm1-7-Pat1 complex plays a key role in mRNA decapping and 3'-end protection and therefore is required for normal mRNA decay rates in vivo. Lsm1 is a key subunit that is critical for the unique RNA binding properties of this complex. We showed earlier that unlike most Sm-like proteins, Lsm1 uniquely requires both its Sm domain and its C-terminal extension to contribute to the function of the Lsm1-7-Pat1 complex and that the C-terminal segment can associate with the rest of the complex and support the function even in trans. The studies presented here identify a set of residues at the very C-terminal end of Lsm1 to be functionally important and suggest that these residues support the function of the Lsm1-7-Pat1 complex by facilitating RNA binding either directly or indirectly.

The Lsm1-7-Pat1 complex promotes viral RNA translation and replication by differential mechanisms.

The Lsm1-7-Pat1 complex binds to the 3' end of cellular mRNAs and promotes 3' end protection and 5'-3' decay. Interestingly, this complex also specifically binds to cis-acting regulatory sequences of viral positive-strand RNA genomes promoting their translation and subsequent recruitment from translation to replication. Yet, how the Lsm1-7-Pat1 complex regulates these two processes remains elusive. Here, we show that Lsm1-7-Pat1 complex acts differentially in these processes. By using a collection of well-characterized lsm1 mutant alleles and a system that allows the replication of Brome mosaic virus (BMV) in yeast we show that the Lsm1-7-Pat1 complex integrity is essential for both, translation and recruitment. However, the intrinsic RNA-binding ability of the complex is only required for translation. Consistent with an RNA-binding-independent function of the Lsm1-7-Pat1 complex on BMV RNA recruitment, we show that the BMV 1a protein, the sole viral protein required for recruitment, interacts with this complex in an RNA-independent manner. Together, these results support a model wherein Lsm1-7-Pat1 complex binds consecutively to BMV RNA regulatory sequences and the 1a protein to promote viral RNA translation and later recruitment out of the host translation machinery to the viral replication complexes.

Pat1 contributes to the RNA binding activity of the Lsm1-7-Pat1 complex.

A major mRNA decay pathway in eukaryotes is initiated by deadenylation followed by decapping of the oligoadenylated mRNAs and subsequent 5'-to-3' exonucleolytic degradation of the capless mRNA. In this pathway, decapping is a rate-limiting step that requires the hetero-octameric Lsm1-7-Pat1 complex to occur at normal rates in vivo. This complex is made up of the seven Sm-like proteins, Lsm1 through Lsm7, and the Pat1 protein. It binds RNA and has a unique binding preference for oligoadenylated RNAs over polyadenylated RNAs. Such binding ability is crucial for its mRNA decay function in vivo. In order to determine the contribution of Pat1 to the function of the Lsm1-7-Pat1 complex, we compared the RNA binding properties of the Lsm1-7 complex purified from pat1Δ cells and purified Pat1 fragments with that of the wild-type Lsm1-7-Pat1 complex. Our studies revealed that both the Lsm1-7 complex and purified Pat1 fragments have very low RNA binding activity and are impaired in the ability to recognize the oligo(A) tail on the RNA. However, reconstitution of the Lsm1-7-Pat1 complex from these components restored these abilities. We also observed that Pat1 directly contacts RNA in the context of the Lsm1-7-Pat1 complex. These studies suggest that the unique RNA binding properties and the mRNA decay function of the Lsm1-7-Pat1 complex involve cooperation of residues from both Pat1 and the Lsm1-7 ring. Finally our studies also revealed that the middle domain of Pat1 is essential for the interaction of Pat1 with the Lsm1-7 complex in vivo.

Both Sm-domain and C-terminal extension of Lsm1 are important for the RNA-binding activity of the Lsm1-7-Pat1 complex.

Lsm proteins are a ubiquitous family of proteins characterized by the Sm-domain. They exist as hexa- or heptameric RNA-binding complexes and carry out RNA-related functions. The Sm-domain is thought to be sufficient for the RNA-binding activity of these proteins. The highly conserved eukaryotic Lsm1 through Lsm7 proteins are part of the cytoplasmic Lsm1-7-Pat1 complex, which is an activator of decapping in the conserved 5'-3' mRNA decay pathway. This complex also protects mRNA 3'-ends from trimming in vivo. Purified Lsm1-7-Pat1 complex is able to bind RNA in vitro and exhibits a unique binding preference for oligoadenylated RNA (over polyadenylated and unadenylated RNA). Lsm1 is a key subunit that determines the RNA-binding properties of this complex. The normal RNA-binding activity of this complex is crucial for mRNA decay and 3'-end protection in vivo and requires the intact Sm-domain of Lsm1. Here, we show that though necessary, the Sm-domain of Lsm1 is not sufficient for the normal RNA-binding ability of the Lsm1-7-Pat1 complex. Deletion of the C-terminal domain (CTD) of Lsm1 (while keeping the Sm-domain intact) impairs mRNA decay in vivo and results in Lsm1-7-Pat1 complexes that are severely impaired in RNA binding in vitro. Interestingly, the mRNA decay and 3'-end protection defects of such CTD-truncated lsm1 mutants could be suppressed in trans by overexpression of the CTD polypeptide. Thus, unlike most Sm-like proteins, Lsm1 uniquely requires both its Sm-domain and CTD for its normal RNA-binding function.

Activation of decapping involves binding of the mRNA and facilitation of the post-binding steps by the Lsm1-7-Pat1 complex.

Decapping is a critical step in the conserved 5'-to-3' mRNA decay pathway of eukaryotes. The hetero-octameric Lsm1-7-Pat1 complex is required for normal rates of decapping in this pathway. This complex also protects the mRNA 3'-ends from trimming in vivo. To elucidate the mechanism of decapping, we analyzed multiple lsm1 mutants, lsm1-6, lsm1-8, lsm1-9, and lsm1-14, all of which are defective in decapping and 3'-end protection but unaffected in Lsm1-7-Pat1 complex integrity. The RNA binding ability of the mutant complex was found to be almost completely lost in the lsm1-8 mutant but only partially impaired in the other mutants. Importantly, overproduction of the Lsm1-9p- or Lsm1-14p-containing (but not Lsm1-8p-containing) mutant complexes in wild-type cells led to a dominant inhibition of mRNA decay. Further, the mRNA 3'-end protection defect of lsm1-9 and lsm1-14 cells, but not the lsm1-8 cells, could be partly suppressed by overproduction of the corresponding mutant complexes in those cells. These results suggest the following: (1) Decapping requires both binding of the Lsm1-7-Pat1 complex to the mRNA and facilitation of the post-binding events, while binding per se is sufficient for 3'-end protection. (2) A major block exists at the post-binding steps in the lsm1-9 and lsm1-14 mutants and at the binding step in the lsm1-8 mutant. Consistent with these ideas, the lsm1-9, 14 allele generated by combining the mutations of lsm1-9 and lsm1-14 alleles had almost fully lost the RNA binding activity of the complex and behaved like the lsm1-8 mutant.

lsm1 mutations impairing the ability of the Lsm1p-7p-Pat1p complex to preferentially bind to oligoadenylated RNA affect mRNA decay in vivo.

The poly(A) tail is a crucial determinant in the control of both mRNA translation and decay. Poly(A) tail length dictates the triggering of the degradation of the message body in the major 5' to 3' and 3' to 5' mRNA decay pathways of eukaryotes. In the 5' to 3' pathway oligoadenylated but not polyadenylated mRNAs are selectively decapped in vivo, allowing their subsequent degradation by 5' to 3' exonucleolysis. The conserved Lsm1p-7p-Pat1p complex is required for normal rates of decapping in vivo, and the purified complex exhibits strong binding preference for oligoadenylated RNAs over polyadenylated or unadenylated RNAs in vitro. In the present study, we show that two lsm1 mutants produce mutant complexes that fail to exhibit such higher affinity for oligoadenylated RNA in vitro. Interestingly, these mutant complexes are normal with regard to their integrity and retain the characteristic RNA binding properties of the wild-type complex, namely, binding near the 3'-end of the RNA, having higher affinity for unadenylated RNAs that carry U-tracts near the 3'-end over those that do not and exhibiting similar affinities for unadenylated and polyadenylated RNAs. Yet, these lsm1 mutants exhibit a strong mRNA decay defect in vivo. These results underscore the importance of Lsm1p-7p-Pat1p complex-mRNA interaction for mRNA decay in vivo and imply that the oligo(A) tail mediated enhancement of such interaction is crucial in that process.

The decapping activator Lsm1p-7p-Pat1p complex has the intrinsic ability to distinguish between oligoadenylated and polyadenylated RNAs.

Decapping is a critical step in mRNA decay. In the 5'-to-3' mRNA decay pathway conserved in all eukaryotes, decay is initiated by poly(A) shortening, and oligoadenylated mRNAs (but not polyadenylated mRNAs) are selectively decapped allowing their subsequent degradation by 5' to 3' exonucleolysis. The highly conserved heptameric Lsm1p-7p complex (made up of the seven Sm-like proteins, Lsm1p-Lsm7p) and its interacting partner Pat1p activate decapping by an unknown mechanism and localize with other decapping factors to the P-bodies in the cytoplasm. The Lsm1p-7p-Pat1p complex also protects the 3'-ends of mRNAs in vivo from trimming, presumably by binding to the 3'-ends. In order to determine the intrinsic RNA-binding properties of this complex, we have purified it from yeast and carried out in vitro analyses. Our studies revealed that it directly binds RNA at/near the 3'-end. Importantly, it possesses the intrinsic ability to distinguish between oligoadenylated and polyadenylated RNAs such that the former are bound with much higher affinity than the latter. These results indicate that the intrinsic RNA-binding characteristics of this complex form a critical determinant of its in vivo interactions and functions.

Src-mediated tyrosine phosphorylation of p47phox in hyperoxia-induced activation of NADPH oxidase and generation of reactive oxygen species in lung endothelial cells.

Superoxide (O(2)(-)) production by nonphagocytes, similar to phagocytes, is by activation of the NADPH oxidase multicomponent system. Although activation of neutrophil NADPH oxidase involves extensive serine phosphorylation of p47(phox), the role of tyrosine phosphorylation of p47(phox) in NADPH oxidase-dependent O(2)(-) production is unclear. We have shown recently that hyperoxia-induced NADPH oxidase activation in human pulmonary artery endothelial cells (HPAECs) is regulated by mitogen-activated protein kinase signal transduction. Here we provided evidence on the role of nonreceptor tyrosine kinase, Src, in hyperoxia-induced tyrosine phosphorylation of p47(phox) and NADPH oxidase activation in HPAECs. Exposure of HPAECs to hyperoxia for 1 h resulted in increased O(2)(-) and reactive oxygen species (ROS) production and enhanced tyrosine phosphorylation of Src as determined by Western blotting with phospho-Src antibodies. Pretreatment of HPAECs with the Src kinase inhibitor PP2 (1 mum) or transient expression of a dominant-negative mutant of Src attenuated hyperoxia-induced tyrosine phosphorylation of Src and ROS production. Furthermore, exposure of cells to hyperoxia enhanced tyrosine phosphorylation of p47(phox) and its translocation to cell peripheries that were attenuated by PP2. In vitro, Src phosphorylated recombinant p47(phox) in a time-dependent manner. Src immunoprecipitates of cell lysates from control cells revealed the presence of immunodetectable p47(phox) and p67(phox), suggesting the association of oxidase components with Src under basal conditions. Moreover, exposure of HPAECs to hyperoxia for 1 h enhanced the association of p47(phox), but not p67(phox), with Src. These results indicated that Src-dependent tyrosine phosphorylation of p47(phox) regulates hyperoxia-induced NADPH oxidase activation and ROS production in HPAECs.

Mutations in the Saccharomyces cerevisiae LSM1 gene that affect mRNA decapping and 3' end protection.

The decapping of eukaryotic mRNAs is a key step in their degradation. The heteroheptameric Lsm1p-7p complex is a general activator of decapping and also functions in protecting the 3' ends of deadenylated mRNAs from a 3'-trimming reaction. Lsm1p is the unique member of the Lsm1p-7p complex, distinguishing that complex from the functionally different Lsm2p-8p complex. To understand the function of Lsm1p, we constructed a series of deletion and point mutations of the LSM1 gene and examined their effects on phenotype. These studies revealed the following: (i) Mutations affecting the predicted RNA-binding and inter-subunit interaction residues of Lsm1p led to impairment of mRNA decay, suggesting that the integrity of the Lsm1p-7p complex and the ability of the Lsm1p-7p complex to interact with mRNA are important for mRNA decay function; (ii) mutations affecting the predicted RNA contact residues did not affect the localization of the Lsm1p-7p complex to the P-bodies; (iii) mRNA 3'-end protection could be indicative of the binding of the Lsm1p-7p complex to the mRNA prior to activation of decapping, since all the mutants defective in mRNA 3' end protection were also blocked in mRNA decay; and (iv) in addition to the Sm domain, the C-terminal domain of Lsm1p is also important for mRNA decay function.

Species differences in TSIX/Tsix reveal the roles of these genes in X-chromosome inactivation.

Transcriptional silencing of the human inactive X chromosome is induced by the XIST gene within the human X-inactivation center. The XIST allele must be turned off on one X chromosome to maintain its activity in cells of both sexes. In the mouse placenta, where X inactivation is imprinted (the paternal X chromosome is always inactive), the maternal Xist allele is repressed by a cis-acting antisense transcript, encoded by the Tsix gene. However, it remains to be seen whether this antisense transcript protects the future active X chromosome during random inactivation in the embryo proper. We recently identified the human TSIX gene and showed that it lacks key regulatory elements needed for the imprinting function of murine Tsix. Now, using RNA FISH for cellular localization of transcripts in human fetal cells, we show that human TSIX antisense transcripts are unable to repress XIST. In fact, TSIX is transcribed only from the inactive X chromosome and is coexpressed with XIST. Also, TSIX is not maternally imprinted in placental tissues, and its transcription persists in placental and fetal tissues, throughout embryogenesis. Therefore, the repression of Xist by mouse Tsix has no counterpart in humans, and TSIX is not the gene that protects the active X chromosome from random inactivation. Because human TSIX cannot imprint X inactivation in the placenta, it serves as a mutant for mouse Tsix, providing insights into features responsible for antisense activity in imprinted X inactivation.