Biochemical characterization of INTS3 and C9ORF80, two subunits of hNABP1/2 heterotrimeric complex in nucleic acid binding.

Human nucleic acid-binding protein 1 and 2 (hNABP1 and hNABP2, also known as hSSB2 and hSSB1 respectively) form two separate and independent complexes with two identical proteins, integrator complex subunit 3 (INTS3) and C9ORF80. We and other groups have demonstrated that hNABP1 and 2 are single-stranded (ss) DNA- and RNA-binding proteins, and function in DNA repair; however, the function of INTS3 and C9OFR80 remains elusive. In the present study, we purified recombinant proteins INTS3 and C9ORF80 to near homogeneity. Both proteins exist as a monomer in solution; however, C9ORF80 exhibits anomalous behavior on SDS-PAGE and gel filtration because of 48% random coil present in the protein. Using electrophoretic mobility shift assay (EMSA), INTS3 displays higher affinity toward ssRNA than ssDNA, and C9ORF80 binds ssDNA but not ssRNA. Neither of them binds dsDNA, dsRNA, or RNA : DNA hybrid. INTS3 requires minimum of 30 nucleotides, whereas C9OFR80 requires 20 nucleotides for its binding, which increased with the increasing length of ssDNA. Interestingly, our GST pulldown results suggest that the N-terminus of INTS3 is involved in protein-protein interaction, while EMSA implies that the C-terminus is required for nucleic acid binding. Furthermore, we purified the INTS3-hNABP1/2-C9ORF80 heterotrimeric complex. It exhibits weaker binding compared with the individual hNABP1/2; interestingly, the hNABP1 complex prefers ssDNA, whereas hNABP2 complex prefers ssRNA. Using reconstituted heterotrimeric complex from individual proteins, EMSA demonstrates that INTS3, but not C9ORF80, affects the nucleic acid-binding ability of hNABP1 and hNABP2, indicating that INTS3 might regulate hNABP1/2's biological function, while the role of C9ORF80 remains unknown.

Insulin-like growth factor-1 signaling in the tumor microenvironment: Carcinogenesis, cancer drug resistance, and therapeutic potential.

The tumor microenvironment fuels tumorigenesis and induces the development of resistance to anticancer drugs. A growing number of reports support that the tumor microenvironment mediates these deleterious effects partly by overexpressing insulin-like growth factor 1 (IGF-1). IGF-1 is known for its role to support cancer progression and metastasis through the promotion of neovascularization in transforming tissues, and the promotion of the proliferation, maintenance and migration of malignant cells. Anti-IGF therapies showed potent anticancer effects and the ability to suppress cancer resistance to various chemotherapy drugs in in vivo and in vitro preclinical studies. However, high toxicity and resistance to these agents are increasingly being reported in clinical trials. We review data supporting the notion that tumor microenvironment mediates tumorigenesis partly through IGF-1 signaling pathway. We also discuss the therapeutic potential of IGF-1 receptor targeting, with special emphasis on the ability of IGF-R silencing to overcome chemotherapy drug resistance, as well as the challenges for clinical use of anti-IGF-1R therapies.

RNA-binding proteins in human genetic disease.

RNA-binding proteins (RBPs) are key components in RNA metabolism, regulating the temporal, spatial and functional dynamics of RNAs. Altering the expression of RBPs has profound implications for cellular physiology, affecting RNA processes from pre-mRNA splicing to protein translation. Recent genetic and proteomic data and evidence from animal models reveal that RBPs are involved in many human diseases ranging from neurologic disorders to cancer. Here we review the emerging evidence showing the involvement of RBPs in many disease networks and conclude that defects in RNA metabolism caused by aberrations in RBPs might underlie a broader spectrum of complex human disorders.

Breast tumor kinase BRK requires kinesin-2 subunit KAP3A in modulation of cell migration.

BReast tumor Kinase (BRK) also known as protein kinase 6 (PTK6) is a nonreceptor tyrosine kinase overexpressed in the majority of human breast tumors. Although some studies have implicated BRK in signalling, cell proliferation and migration, the precise intracellular role of BRK has not been fully elucidated. The RNA-binding protein Sam68, and adaptor proteins paxillin and STAT3 are the only BRK substrates that link BRK to signal transduction. To identify new BRK substrates, we screened high-density protein filter arrays by large-scale in vitro kinase assays using active recombinant BRK. We identified at least 4 BRK targets comprising the alpha-subunit of stimulatory guanine nucleotide binding protein (GNAS), FL139441, beta-tubulin and kinesin associated protein 3A (KAP3A) and validated them as BRK substrates using a secondary assay. Further characterization revealed that KAP3A is an in vivo substrate of BRK and associates with BRK in breast cancer cells. We show that BRK specifically phosphorylated tyrosine residues at the C-terminus of KAP3A and induces delocalization of KAP3A from punctate nuclear localization to a diffuse nucleo-cytoplasmic pattern. Functionally, we demonstrate that KAP3A knockdown results in suppression of BRK-induced migration of breast cancer cells and show that the C-terminal deletion mutant of KAP3A acts as a dominant negative in BRK-induced cell migration. Our findings therefore reveal new substrates of BRK and define KAP3A as a physiological substrate of BRK during cell migration.

Motor coordination defects in mice deficient for the Sam68 RNA-binding protein.

The role of RNA-binding proteins in the central nervous system and more specifically their role in motor coordination and learning are poorly understood. We previously reported that ablation of RNA-binding protein Sam68 in mice results in male sterility and delayed mammary gland development and protection against osteoporosis in females. Sam68 however is highly expressed in most regions of the brain especially the cerebellum and thus we investigated the cerebellar-related manifestations in Sam68-null mice. We analyzed the mice for motor function, sensory function, and learning and memory abilities. Herein, we report that Sam68-null mice have motor coordination defects as assessed by beam walking and rotorod performance. Forty-week-old Sam68-null mice (n=12) were compared to their wild-type littermates (n=12). The Sam68-null mice exhibited more hindpaw faults in beam walking tests and fell from the rotating drum at lower speeds and prematurely compared to the wild-type controls. The Sam68-null mice were, however, normal for forelimb strength, tail-hang reflex, balance test, grid walking, the Morris water task, recognition memory, visual discrimination, auditory stimulation and conditional taste aversion. Our findings support a role for Sam68 in the central nervous system in the regulation of motor coordination.

Repression of Fyn-related kinase in breast cancer cells is associated with promoter site-specific CpG methylation.

The triple-negative breast cancer subtype is highly aggressive and has no defined therapeutic target. Fyn-related kinase (FRK) is a non-receptor tyrosine kinase, reported to be downregulated in breast cancer and gliomas, where it is suggested to have tumor suppressor activity. We examined the expression profile of FRK in a panel of 40 breast cancer cells representing all the major subtypes, as well as in 4 non-malignant mammary epithelial cell lines. We found that FRK expression was significantly repressed in a proportion of basal B breast cancer cell lines. We then determined the mechanism of suppression of FRK in FRK-low or negative cell lines. In silico analyses of the FRK promoter region led to the identification of at least 17 CpG sites. Bisulphite sequencing of the promoter region revealed that two of these sites were consistently methylated in FRK-low/negative cell lines and especially in the basal B breast cancer subtype. We further show that treatment of these cells with histone deacetylase inhibitors, Entinostat and Mocetinostat' promoted re-expression of FRK mRNA and protein. Further, using luciferase reporter assays, we show that both GATA3-binding protein FOG1 and constitutively active STAT5A increased the activity of FRK promoter. Together, our results present the first evidence that site-specific promoter methylation contributes to the repression of FRK more so in basal B breast cancers. Our study also highlights the potential clinical significance of targeting FRK using epigenetic drugs specifically in basal B breast cancers which are usually triple negative and very aggressive.

Advancement of mass spectrometry-based proteomics technologies to explore triple negative breast cancer.

Understanding the complexity of cancer biology requires extensive information about the cancer proteome over the course of the disease. The recent advances in mass spectrometry-based proteomics technologies have led to the accumulation of an incredible amount of such proteomic information. This information allows us to identify protein signatures or protein biomarkers, which can be used to improve cancer diagnosis, prognosis and treatment. For example, mass spectrometry-based proteomics has been used in breast cancer research for over two decades to elucidate protein function. Breast cancer is a heterogeneous group of diseases with distinct molecular features that are reflected in tumour characteristics and clinical outcomes. Compared with all other subtypes of breast cancer, triple-negative breast cancer is perhaps the most distinct in nature and heterogeneity. In this review, we provide an introductory overview of the application of advanced proteomic technologies to triple-negative breast cancer research.

Sam68, the KH domain-containing superSTAR.

Sam68 is one of the most studied members of the STAR family of RNA-binding proteins since its identification over a decade ago. Since its ascension into prominence, enormous progress has been made to unmask the link between the RNA-binding properties of Sam68 and the regulation of cellular processes including signal transduction, cell cycle regulation and tumorigenesis and RNA biogenesis in general. In this review we provide a detailed description of the functional domains of Sam68 and the possible biological roles that justify its superSTAR status.

BRK targets Dok1 for ubiquitin-mediated proteasomal degradation to promote cell proliferation and migration.

Breast tumor kinase (BRK), also known as protein tyrosine kinase 6 (PTK6), is a non-receptor tyrosine kinase overexpressed in more that 60% of human breast carcinomas. The overexpression of BRK has been shown to sensitize mammary epithelial cells to mitogenic signaling and to promote cell proliferation and tumor formation. The molecular mechanisms of BRK have been unveiled by the identification and characterization of BRK target proteins. Downstream of tyrosine kinases 1 or Dok1 is a scaffolding protein and a substrate of several tyrosine kinases. Herein we show that BRK interacts with and phosphorylates Dok1 specifically on Y362. We demonstrate that this phosphorylation by BRK significantly downregulates Dok1 in a ubiquitin-proteasome-mediated mechanism. Together, these results suggest a novel mechanism of action of BRK in the promotion of tumor formation, which involves the targeting of tumor suppressor Dok1 for degradation through the ubiquitin proteasomal pathway.

The unique N-terminal region of SRMS regulates enzymatic activity and phosphorylation of its novel substrate docking protein 1.

SRMS (Src-related tyrosine kinase lacking C-terminal regulatory tyrosine and N-terminal myristoylation sites) belongs to a family of nonreceptor tyrosine kinases, which also includes breast tumour kinase and Fyn-related kinase. SRMS, similar to breast tumour kinase and Fyn-related kinase, harbours a Src homology 3 and Src homology 2, as well as a protein kinase domain. However, unlike breast tumour kinase and Fyn-related kinase, SRMS lacks a C-terminal regulatory tail but distinctively possesses an extended N-terminal region. Both breast tumour kinase and Fyn-related kinase play opposing roles in cell proliferation and signalling. SRMS, however, is an understudied member of this family. Although cloned in 1994, information on the biochemical, cellular and physiological roles of SRMS remains unreported. The present study is the first to explore the expression pattern of SRMS in breast cancers, its enzymatic activity and autoregulatory elements, and the characterization of docking protein 1 as its first bonafide substrate. We found that, similar to breast tumour kinase, SRMS is highly expressed in most breast cancers compared to normal mammary cell lines and tissues. We generated a series of SRMS point and deletion mutants and assessed enzymatic activity, subcellular localization and substrate recognition. We report for the first time that ectopically-expressed SRMS is constitutively active and that its N-terminal region regulates the enzymatic activity of the protein. Finally, we present evidence indicating that docking protein 1 is a direct substrate of SRMS. Our data demonstrate that, unlike members of the Src family, the enzymatic activity of SRMS is regulated by the intramolecular interactions involving the N-terminus of the enzyme and that docking protein 1 is a bona fide substrate of SRMS.

BRK phosphorylates PSF promoting its cytoplasmic localization and cell cycle arrest.

BReast tumor Kinase (BRK) also known as protein kinase 6 (PTK6) is a non-receptor tyrosine kinase overexpressed in the majority of human breast carcinoma. The expression of BRK is a known prognostic marker of breast carcinoma. BRK has been shown to lie downstream of epidermal growth factor (EGF) signaling and mediate effects on cell proliferation and migration. To identify BRK substrates and interacting proteins, we undertook a proteomic approach. BRK immune complexes were purified from the BT-20 breast cancer cell line and analyzed by mass spectrometry. Herein, we report the identification of PSF, the polypyrimidine tract-binding (PTB) protein-associated splicing factor, as a BRK-interacting protein and substrate. BRK and PSF co-eluted in a large protein complex that was regulated by EGF stimulation. Furthermore, BRK and PSF co-immunoprecipitated in BT-20 cells and we defined the interaction as being an SH3 domain-polyproline interaction. The C-terminal tyrosines of PSF were the site of phosphorylation by BRK. Moreover, tyrosine phosphorylation of PSF was also observed upon EGF stimulation, consistent with a role of PSF and BRK downstream of the EGF receptor. Interestingly, the tyrosine phosphorylation promoted the cytoplasmic relocalization of PSF, impaired its binding to polypyrimidine RNA, and led to cell cycle arrest. Our findings show that BRK targets the PSF RNA-binding protein during EGF stimulation.

Targeting the RNA-binding protein Sam68 as a treatment for cancer?

The contradictory properties of RNA-binding proteins (RBPs) have mystified their roles in human diseases including cancer. Are certain RBPs oncogenes or tumor suppressors? In the case of the signal transduction activator of RNA metabolism (STAR) family of hnRNP K homology (KH)-domain-containing RBPs, the dominant view with loose experimental evidence is that these proteins are tumor suppressors. However, recent developments support a pro-oncogenic role for archetypical STAR protein Sam68. Sam68-null mice are not prone to cancer, but instead display pronounced defects in mammary gland ductal development, and haploinsufficiency of Sam68 impedes mammary tumor onset and tumor multiplicity in mouse models expressing the mammary-targeted polyoma middle T antigen oncogene. These advances have increased the interest in the role of Sam68 as a positive regulator of cancer progression and position Sam68 as a viable therapeutic target. Retrospective and perspective implications of Sam68 in cancer are discussed.