PRMT7 methylates eukaryotic translation initiation factor 2α and regulates its role in stress granule formation.

Protein arginine methyltransferases (PRMTs) are a family of enzymes that modify proteins by methylating the guanidino nitrogen atoms of arginine residues to regulate cellular processes such as chromatin remodeling, pre-mRNA splicing, and signal transduction. PRMT7 is the single type III PRMT solely capable of arginine monomethylation. To date, other than histone proteins, there are very few identified substrates of PRMT7. We therefore performed quantitative mass spectrometry experiments to identify PRMT7's interactome and potential substrates to better characterize the enzyme's biological function(s) in cells. These experiments revealed that PRMT7 interacts with and can methylate eukaryotic translation initiation factor 2 alpha (eIF2α), in vitro and in breast cancer cells. Furthermore, we uncovered a potential regulatory interplay between eIF2α arginine methylation by PRMT7 and stress-induced phosphorylation status of eIF2α at serine 51. Finally, we demonstrated that PRMT7 is required for eIF2α-dependent stress granule formation in the face of various cellular stresses. Altogether, our findings implicate PRMT7 as a novel mediator of eIF2α-dependent cellular stress response pathways.

Atg5 Disassociates the V1V0-ATPase to Promote Exosome Production and Tumor Metastasis Independent of Canonical Macroautophagy.

Autophagy and autophagy-related genes (Atg) have been attributed prominent roles in tumorigenesis, tumor growth, and metastasis. Extracellular vesicles called exosomes are also implicated in cancer metastasis. Here, we demonstrate that exosome production is strongly reduced in cells lacking Atg5 and Atg16L1, but this is independent of Atg7 and canonical autophagy. Atg5 specifically decreases acidification of late endosomes where exosomes are produced, disrupting the acidifying V1V0-ATPase by removing a regulatory component, ATP6V1E1, into exosomes. The effect of Atg5 on exosome production promotes the migration and in vivo metastasis of orthotopic breast cancer cells. These findings uncover mechanisms controlling exosome release and identify means by which autophagy-related genes can contribute to metastasis in autophagy-independent pathways.

RNA binding protein RALY promotes Protein Arginine Methyltransferase 1 alternatively spliced isoform v2 relative expression and metastatic potential in breast cancer cells.

Aberrant expression of Protein Arginine Methyltransferases (PRMTs) has been observed in several cancer types, including breast cancer. We previously reported that the PRMT1v2 isoform, which is generated through inclusion of alternative exon 2, is overexpressed in breast cancer cells and promotes their invasiveness. However, the precise mechanism by which expression of this isoform is controlled and how it is dysregulated in breast cancer remains unknown. Using a custom RNA interference-based screen, we identified several RNA binding proteins (RBP) which, when knocked down, altered the relative abundance of the alternatively spliced PRMT1v2 isoform. Amongst the top hits were SNW Domain containing 1 (SNW1) and RBP-associated with lethal yellow mutation (RALY), which both associated with the PRMT1 pre-mRNA and upon depletion caused an increase or decrease in the relative abundance of PRMT1v2 isoform mRNA and protein. Most importantly, a significant decrease in invasion was observed upon RALY knockdown in aggressive breast cancer cells, consistent with targeting PRMT1v2 directly, and this effect was rescued by the exogenous re-expression of PRMT1v2. We show that SNW1 expression is decreased, while RALY expression is increased in breast cancer cells and tumours, which correlates with decreased patient survival. This work revealed crucial insight into the mechanisms regulating the expression of the PRMT1 alternatively spliced isoform v2 and its dysregulation in breast cancer. It also provides proof-of-concept support for the development of therapeutic strategies where regulators of PRMT1 exon 2 alternative splicing are targeted as an approach to selectively reduce PRMT1v2 levels and metastasis in breast cancer.

Tudor Domain Containing Protein 3 Promotes Tumorigenesis and Invasive Capacity of Breast Cancer Cells.

Tudor domain containing protein 3 (TDRD3) is a modular protein identified based on its ability to recognize methylated arginine motifs through its Tudor domain. We have previously shown that TDRD3 localizes to cytoplasmic stress granules, a structure shown to promote survival upon treatment with chemotherapeutic drugs in cancer cells. Here, we report TDRD3 as a novel regulator of cell proliferation and invasion in breast cancer cells. Our study also demonstrates that TDRD3 depletion inhibits tumor formation and metastasis to the lung in vivo. Furthermore, we show that TDRD3 regulates the expression of a number of key genes associated with promotion of breast cancer tumorigenesis and disease progression. Strikingly, we report that TDRD3 regulates some of these key targets at the level of translation. These findings provide the first experimental demonstration of a functional role for TDRD3 in promoting breast cancer development and progression, and identify TDRD3 as a potential new therapeutic target for breast cancer.

Arginine methyltransferases as novel therapeutic targets for breast cancer.

Breast cancer is the most commonly diagnosed female cancer in the world. Though therapeutic treatments are available to treat breast cancer and in some instances are successful, the occurrence of unsuccessful treatment, or the rate of tumour recurrence, still remains strikingly high. Therefore, novel therapeutic treatment targets need to be discovered and tested. The protein arginine methyltransferases (PRMTs) are a family of enzymes that catalyse arginine methylation and are implicated in a myriad of cellular pathways including transcription, DNA repair, RNA metabolism, signal transduction, protein-protein interactions and subcellular localisation. In breast cancer, the expression levels and enzymatic activity of a number of PRMTs is dysregulated; significantly altering the regulation of many cellular pathways that are implicated in breast cancer development and progression. Here, we review the current knowledge on PRMTs in breast cancer and provide a rationale for how PRMTs may provide novel therapeutic targets for the treatment of breast cancer.

Identification of the PRMT1v1 and PRMT1v2 specific interactomes by quantitative mass spectrometry in breast cancer cells.

Arginine methylation is catalyzed by a family of enzymes called protein arginine methyltransferases (PRMTs). The PRMT1 gene generates at least seven distinct alternatively spliced isoforms (PRMT v1-v7), which together contribute a significant portion of the cellular arginine methylome. The distinct biochemical and biological functions of these PRMT1 isoforms have not been well characterized. Previously we have shown that while both PRMT1v1 and PRMT1v2 are overexpressed in breast cancer cells, PRMT1v2 specifically promotes breast cancer cell survival and invasion. These isoforms also have distinct subcellular localizations, PRMT1v1 is mainly nuclear and PRMT1v2 cytosolic. To gain further knowledge into their isoform-specific roles within cells we used a SILAC-based quantitative affinity purification/MS approach to identify their individual protein interactomes in breast cancer cells. This analysis has uncovered distinct interactomes for PRMT1v1 and PRMT1v2. Consistent with their distinct subcellular localizations, PRMT1v1 enriched a mainly nuclear protein interactome, while PRMT1v2 enriched predominantly cytoplasmic interactors from whole-cell extracts. Furthermore, these interactomes revealed that PRMT1v1 has a role in regulating gene expression, while PRMT1v2 functions in cytoskeletal dynamics. These results highlight the unique functions of these isoforms and the distinct roles they may play within cells, with potential implications for breast cancer and other diseases.

Protein arginine methyltransferase 7 promotes breast cancer cell invasion through the induction of MMP9 expression.

Recent evidence points to the protein arginine methyltransferase (PRMT) family of enzymes playing critical roles in cancer. PRMT7 has been identified in several gene expression studies to be associated with increased metastasis and decreased survival in breast cancer patients. However, this has not been extensively studied. Here we report that PRMT7 expression is significantly upregulated in both primary breast tumour tissues and in breast cancer lymph node metastases. We have demonstrated that reducing PRMT7 levels in invasive breast cancer cells using RNA interference significantly decreased cell invasion in vitro and metastasis in vivo. Conversely, overexpression of PRMT7 in non-aggressive MCF7 cells enhanced their invasiveness. Furthermore, we show that PRMT7 induces the expression of matrix metalloproteinase 9 (MMP9), a well-known mediator of breast cancer metastasis. Importantly, we significantly rescued invasion of aggressive breast cancer cells depleted of PRMT7 by the exogenous expression of MMP9. Our results demonstrate that upregulation of PRMT7 in breast cancer may have a significant role in promoting cell invasion through the regulation of MMP9. This identifies PRMT7 as a novel and potentially significant biomarker and therapeutic target for breast cancer.

Role of PRMTs in cancer: Could minor isoforms be leaving a mark?

Protein arginine methyltransferases (PRMTs) catalyze the methylation of a variety of protein substrates, many of which have been linked to the development, progression and aggressiveness of different types of cancer. Moreover, aberrant expression of PRMTs has been observed in several cancer types. While the link between PRMTs and cancer is a relatively new area of interest, the functional implications documented thus far warrant further investigations into its therapeutic potential. However, the expression of these enzymes and the regulation of their activity in cancer are still significantly understudied. Currently there are nine main members of the PRMT family. Further, the existence of alternatively spliced isoforms for several of these family members provides an additional layer of complexity. Specifically, PRMT1, PRMT2, CARM1 and PRMT7 have been shown to have alternative isoforms and others may be currently unrealized. Our knowledge with respect to the relative expression and the specific functions of these isoforms is largely lacking and needs attention. Here we present a review of the current knowledge of the known alternative PRMT isoforms and provide a rationale for how they may impact on cancer and represent potentially useful targets for the development of novel therapeutic strategies.

Alternatively spliced protein arginine methyltransferase 1 isoform PRMT1v2 promotes the survival and invasiveness of breast cancer cells.

Protein arginine methylation is catalyzed by protein arginine methyltransferases (PRMTs) and plays an important role in many cellular processes. Aberrant PRMT expression has been observed in several common cancer types; however, their precise contribution to the cell transformation process is not well understood. We previously reported that the PRMT1 gene generates several alternatively spliced isoforms, and our initial biochemical characterization of these isoforms revealed that they exhibit distinct substrate specificity and subcellular localization. We focus here on the PRMT1v2 isoform, which is the only predominantly cytoplasmic isoform, and we have found that its relative expression is increased in breast cancer cell lines and tumors. Specific depletion of PRMT1v2 using RNA interference caused a significant decrease in cancer cell survival due to an induction of apoptosis. Furthermore, depletion of PRMT1v2 in an aggressive cancer cell line significantly decreased cell invasion. We also demonstrate that PRMT1v2 overexpression in a non-aggressive cancer cell line was sufficient to render them more invasive. Importantly, this novel activity is specific to PRMT1v2, as overexpression of other isoforms did not enhance invasion. Moreover, this activity requires both proper subcellular localization and methylase activity. Lastly, PRMT1v2 overexpression altered cell morphology and reduced cell-cell adhesion, a phenomenon that we convincingly linked with reduced ß-catenin protein expression. Overall, we demonstrate a specific role for PRMT1v2 in breast cancer cell survival and invasion, underscoring the importance of identifying and characterizing the distinct functional differences between PRMT1 isoforms.

Coordination of glioblastoma cell motility by PKCι.

BACKGROUND: Glioblastoma is one of the deadliest forms of cancer, in part because of its highly invasive nature. The tumor suppressor PTEN is frequently mutated in glioblastoma and is known to contribute to the invasive phenotype. However the downstream events that promote invasion are not fully understood. PTEN loss leads to activation of the atypical protein kinase C, PKCι. We have previously shown that PKCι is required for glioblastoma cell invasion, primarily by enhancing cell motility. Here we have used time-lapse videomicroscopy to more precisely define the role of PKCι in glioblastoma. RESULTS: Glioblastoma cells in which PKCι was either depleted by shRNA or inhibited pharmacologically were unable to coordinate the formation of a single leading edge lamellipod. Instead, some cells generated multiple small, short-lived protrusions while others generated a diffuse leading edge that formed around the entire circumference of the cell. Confocal microscopy showed that this behavior was associated with altered behavior of the cytoskeletal protein Lgl, which is known to be inactivated by PKCι phosphorylation. Lgl in control cells localized to the lamellipod leading edge and did not associate with its binding partner non-muscle myosin II, consistent with it being in an inactive state. In PKCι-depleted cells, Lgl was concentrated at multiple sites at the periphery of the cell and remained in association with non-muscle myosin II. Videomicroscopy also identified a novel role for PKCι in the cell cycle. Cells in which PKCι was either depleted by shRNA or inhibited pharmacologically entered mitosis normally, but showed marked delays in completing mitosis. CONCLUSIONS: PKCι promotes glioblastoma motility by coordinating the formation of a single leading edge lamellipod and has a role in remodeling the cytoskeleton at the lamellipod leading edge, promoting the dissociation of Lgl from non-muscle myosin II. In addition PKCι is required for the transition of glioblastoma cells through mitosis. PKCι therefore has a role in both glioblastoma invasion and proliferation, two key aspects in the malignant nature of this disease.

Activation of p38MAPK contributes to expanded polyglutamine-induced cytotoxicity.

BACKGROUND: The signaling pathways that may modulate the pathogenesis of diseases induced by expanded polyglutamine proteins are not well understood. METHODOLOGIES/PRINCIPAL FINDINGS: Herein we demonstrate that expanded polyglutamine protein cytotoxicity is mediated primarily through activation of p38MAPK and that the atypical PKC iota (PKCiota) enzyme antagonizes polyglutamine-induced cell death through induction of the ERK signaling pathway. We show that pharmacological blockade of p38MAPK rescues cells from polyglutamine-induced cell death whereas inhibition of ERK recapitulates the sensitivity observed in cells depleted of PKCiota by RNA interference. We provide evidence that two unrelated proteins with expanded polyglutamine repeats induce p38MAPK in cultured cells, and demonstrate induction of p38MAPK in an in vivo model of neurodegeneration (spinocerebellar ataxia 1, or SCA-1). CONCLUSIONS/SIGNIFICANCE: Taken together, our data implicate activated p38MAPK in disease progression and suggest that its inhibition may represent a rational strategy for therapeutic intervention in the polyglutamine disorders.

Transgenic overexpression of IGF-II induces spontaneous lung tumors: a model for human lung adenocarcinoma.

Elevated levels of insulin-like growth factor (IGF)-II are associated with a poor prognosis in human pulmonary adenocarcinoma; however, a causal role for IGF-II in pulmonary adenocarcinoma has not been demonstrated. Here, we show that transgenic overexpression of IGF-II in lung epithelium induces lung tumors in 69% of mice older than 18 months of age. These tumors displayed morphological characteristics of human pulmonary adenocarcinoma such as their epithelial origin, tubulo-acinar architecture and expression of TTF-1, SP-B and proSP-C. Examination of signaling molecules downstream of the IGF-IR showed the activation of either the Erk1/Erk2 or p38 MAPK pathways, but not both, within the lung tumors. Notably, all lung tumors contained high levels of phosphorylated CREB, suggesting that both the Erk1/Erk2 and p38 MAPK pathways converged on this transcription factor. Moreover, IGF-II induced proliferation and CREB phosphorylation in human lung cancer cell lines, suggesting that IGF-II and CREB also contribute to the growth of human lung tumors. Thus, IGF-II is an important genetic factor in the development of lung tumorigenesis, in which activation of CREB is a ubiquitous event. The MMTV-IGF-II transgenic mice provide a critical model for elucidating the role of IGF-II in this fatal human disease.