Slit-Robo signaling induces malignant transformation through Hakai-mediated E-cadherin degradation during colorectal epithelial cell carcinogenesis

The Slit family of guidance cues binds to Roundabout (Robo) receptors and modulates cell migration. We report here that ectopic expression of Slit2 and Robo1 or recombinant Slit2 treatment of Robo1-expressing colorectal epithelial carcinoma cells recruited an ubiquitin ligase Hakai for E-cadherin (E-cad) ubiquitination and lysosomal degradation, epithelial-mesenchymal transition (EMT), and tumor growth and liver metastasis, which were rescued by knockdown of Hakai. In contrast, knockdown of endogenous Robo1 or specific blockade of Slit2 binding to Robo1 prevented E-cad degradation and reversed EMT, resulting in diminished tumor growth and liver metastasis. Ectopic expression of Robo1 also triggered a malignant transformation in Slit2-positive human embryonic kidney 293 cells. Importantly, the expression of Slit2 and Robo1 was significantly associated with an increased metastatic risk and poorer overall survival in colorectal carcinoma patients. We conclude that engagement of Robo1 by Slit2 induces malignant transformation through Hakai-mediated E-cad ubiquitination and lysosomal degradation during colorectal epithelial cell carcinogenesis.     

The Non-Coding RNA Llme23 Drives the Malignant Property of Human Melanoma Cells

Several lines of evidence support the notion that increased RNA-binding ability of polypyrimidine tract-binding (PTB) protein-associated splicing factor (PSF) and aberrant expression of long non-coding RNAs (lncRNAs) are associated with mouse and human tumors. To identify the PSF-binding lncRNA involved in human oncogenesis, we screened a nuclear RNA repertoire of human melanoma cell line, YUSAC, through RNA-SELEX affinity chromatography. A previously unreported lncRNA, termed as Llme23, was found to bind immobilized PSF resin. The specific binding of Llme23 to both recombinant and native PSF protein was confirmed in vitro and in vivo. The expression of PSF-binding Llme23 is exclusively detected in human melanoma lines. Knocking down Llme23 remarkably suppressed the malignant property of YUSAC cells, accompanied by the repressed expression of proto-oncogene Rab23. These results may indicate that Llme23 can function as an oncogenic RNA and directly associate the PSF-binding lncRNA with human melanoma.     

Thioredoxin reductase 1 deficiency reverses tumor phenotype and tumorigenicity of lung carcinoma cells

Dietary selenium has potent cancer prevention activity. Both low molecular weight selenocompounds and selenoproteins are implicated in this effect. Thioredoxin reductase 1 (TR1) is one of the major antioxidant and redox regulators in mammals that supports p53 function and other tumor suppressor activities. However, this selenium-containing oxidoreductase is also overexpressed in many malignant cells and has been proposed as a target for cancer therapy. To further assess the role of TR1 in the malignancy process, we used RNA interference technology to decrease its expression in mouse lung carcinoma (LLC1) cells. Stable transfection of LLC1 cells with a small interfering RNA construct that specifically targets TR1 removal manifested a reversal in the morphology and anchorage-independent growth properties of these cancer cells that made them similar to those of normal cells. The expression of at least two cancer-related protein mRNAs, Hgf and Opn1, were reduced dramatically in the TR1 knockdown cells. Mice injected with the TR1 knockdown showed a dramatic reduction in tumor progression and metastasis compared with those mice injected with the corresponding control vector. In addition, tumors that arose from injected TR1 knockdown cells lost the targeting construct, suggesting that TR1 is essential for tumor growth in mice. These observations provide direct evidence that the reduction of TR1 levels in malignant cells is antitumorigenic and suggest that the enzyme is a prime target for cancer therapy.     

Posttranscriptional Orchestration of an Anti-Apoptotic Program by HuR

The RNA-binding protein HuR can stabilize and/or regulate the translation of target mRNAs, thereby affecting the cellular responses to immune, proliferative, and damaging agents. Here, we discuss emerging evidence that HuR elicits a broad anti-apoptotic function through its influence on the expression of multiple target mRNAs. HuR was previously shown to bind to the mRNA encoding the apoptosome inhibitor prothymosin α (ProTα) and enhanced its translation and cytoplasmic abundance. More recently, HuR was shown to increase the stability of a target mRNA encoding the pro-survival deacetylase SIRT1. The discovery that HuR likewise binds to and promotes the expression of mRNAs encoding Bcl-2 and Mcl-1, two major anti-apoptotic effectors, strongly supports HuR’s role as a key upstream coordinator of a constitutive pro-survival program.     

Visinin-like protein (VILIP) is a neuron-specific calcium-dependent double-stranded RNA-binding protein.

Double-stranded RNA-binding proteins function in regulating the stability, translation, and localization of specific mRNAs. In this study, we have demonstrated that the neuron-specific, calcium-binding protein, visinin-like protein (VILIP) contains one double-stranded RNA-binding domain, a protein motif conserved among many double-stranded RNA-binding proteins. We showed that VILIP can specifically bind double-stranded RNA, and this interaction specifically requires the presence of calcium. Mobility shift studies indicated that VILIP binds double-stranded RNA as a single protein-RNA complex with an apparent equilibrium dissociation constant of 9.0 x 10(-6) M. To our knowledge, VILIP is the first double-stranded RNA-binding protein shown to be calcium-dependent. Furthermore, VILIP specifically binds the 3'-untranslated region of the neurotrophin receptor, trkB, an mRNA localized to hippocampal dendrites in an activity-dependent manner. Given that VILIP is also expressed in the hippocampus, these data suggest that VILIP may employ a novel, calcium-dependent mechanism to regulate its binding to important localized mRNAs in the central nervous system.     

Coordinated control of lumen size and collective migration in the salivary gland

Drosophila Smaug is a sequence-specific RNA-binding protein that can repress the translation and induce the degradation of target mRNAs in the early Drosophila embryo. Our recent work has uncovered a new mechanism of Smaug-mediated translational repression whereby it interacts with and recruits the Argonaute 1 (Ago1) protein to an mRNA. Argonaute proteins are typically recruited to mRNAs through an associated small RNA, such as a microRNA (miRNA). Surprisingly, we found that Smaug is able to recruit Ago1 to an mRNA in a miRNA-independent manner. This work suggests that other RNA-binding proteins are likely to employ a similar mechanism of miRNA-independent Ago recruitment to control mRNA expression. Our work also adds yet another mechanism to the list that Smaug can use to regulate its targets and here we discuss some of the issues that are raised by Smaug’s multi-functional nature.     

Interplay between microRNAs and RNA-binding proteins determines developmental processes

MicroRNAs (miRNAs) are genes involved in normal development and cancer. They inhibit gene expression by associating with 3'-Untranslated regions (3' UTRs) of messenger RNAs (mRNAs), and are thought to regulate a large proportion of protein coding genes. However, it is becoming apparent that miRNA activity is not necessarily always determined by its expression in the cell. MiRNA activity can be affected by RNA-binding proteins (RBPs). For example, the RNA-binding protein HuR associates with the 3'UTR of the CAT1 mRNA after stress, counteracting the effect of miR-122. Second, we found that the expression of an RNA-binding protein called Dead end (Dnd1) prohibits the function of several miRNAs by blocking the accessibility of target mRNAs. Dnd1 function is essential for proper development of primordial germ cells (PGCs) in zebrafish and mammals, indicating a crucial role for RBP/miRNA interplay on 3'UTRs of mRNAs in developmental decisions. In this perspective we discuss the interplay between RBPs and miRNAs in the context of germ cells and review current observations implicating RBPs in miRNA function.     

Inhibiting autophagy by shRNA knockdown

A glance through Autophagy or any other journal in this field shows that it is very common to block autophagy by RNA interference-based knockdown of ATG mRNAs in mammalian cell lines. Our lab’s experience is that this approach can easily make for failed experiments because good knockdown of even essential autophagy regulators does not necessarily mean you will get good inhibition of autophagy, and, over time, cells can find ways to circumvent the inhibitory effects of the knockdown.     

An inducible cytoplasmic factor (AU-B) binds selectively to AUUUA multimers in the 3'' untranslated region of lymphokine mRNA.

Considerable evidence suggests that the metabolism of lymphokine mRNAs can be selectively regulated within the cytoplasm. However, little is known about the mechanism(s) that cells use to discriminate lymphokine mRNAs from other mRNAs within the cytoplasm. In this study we report a sequence-specific cytoplasmic factor (AU-B) that binds specifically to AUUUA multimers present in the 3' untranslated region of lymphokine mRNAs. AU-B does not bind to monomeric AUUUA motifs nor to other AU-rich sequences present in the 3' untranslated region of c-myc mRNA. AU-B RNA-binding activity is not present in quiescent T cells but is rapidly induced by stimulation of the T-cell receptor/CD3 complex. Induction of AU-B RNA-binding activity requires new RNA and protein synthesis. Stabilization of lymphokine mRNA induced by costimulation with phorbol myristate acetate correlates inversely with binding by AU-B. Together, these data suggest that AU-B is a cytoplasmic regulator of lymphokine mRNA metabolism.     

PTB-Associated Splicing Factor (PSF) Is a PPARγ-Binding Protein and Growth Regulator of Colon Cancer Cells

Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor that plays an essential role in cell proliferation, apoptosis, and inflammation. It is over-expressed in many types of cancer, including colon, stomach, breast, and lung cancer, suggesting that regulation of PPARγ might affect cancer pathogenesis. Here, using a proteomic approach, we identify PTB-associated splicing factor (PSF) as a novel PPARγ-interacting protein and demonstrate that PSF is involved in several important regulatory steps of colon cancer cell proliferation. To investigate the relationship between PSF and PPARγ in colon cancer, we evaluated the effects of PSF expression in DLD-1 and HT-29 colon cancer cell lines, which express low and high levels of PPARγ, respectively PSF affected the ability of PPARγ to bind, and expression of PSF siRNA significantly suppressed the proliferation of colon cancer cells. Furthermore, PSF knockdown induced apoptosis via activation of caspase-3. Interestingly, DLD-1 cells were more susceptible to PSF knockdown-induced cell death than HT-29 cells. Our data suggest that PSF is an important regulator of cell death that plays critical roles in the survival and growth of colon cancer cells. The PSF-PPARγ axis may play a role in the control of colorectal carcinogenesis. Taken together, this study is the first to describe the effects of PSF on cell proliferation, tumor growth, and cell signaling associated with PPARγ.     

PSF and p54nrb bind a conserved stem in U5 snRNA

PTB-associated splicing factor (PSF) has been implicated in both early and late steps of pre-mRNA splicing, but its exact role in this process remains unclear. Here we show that PSF interacts with p54nrb, a highly related protein first identified based on cross-reactivity to antibodies against the yeast second-step splicing factor Prpl8. We performed RNA-binding experiments to determine the preferred RNA-binding sequences for PSF and p54nrb, both individually and in combination. In all cases, iterative selection assays identified a purine-rich sequence located on the 3' side of U5 snRNA stem 1b. Filter-binding assays and RNA affinity selection experiments demonstrated that PSF and p54nrb bind U5 snRNA with both the sequence and structure of stem 1b contributing to binding specificity. Sedimentation analyses show that both proteins associate with spliceosomes and with U4/U6.U5 tri-snPNP.     

Sqd interacts with the Drosophila retinoblastoma tumor suppressor Rbf

The retinoblastoma tumor suppressor (RB) serves as a scaffold to coordinate binding of numerous proteins, including E2F and histone deacetylases, through its C-terminal domain. The amino-terminal half of RB has few known binding partners and its function is not well understood. We used the amino-terminal domain of the Drosophila retinoblastoma tumor suppressor Rbf (RbfN) to identify novel binding partners by immunoprecipitation coupled with mass spectrometry. Our experiment revealed that the RNA-binding protein Squid (Sqd) is a putative interacting partner of RbfN. Western blot confirmed that Sqd interacts with the amino-terminal domain of Rbf. We observed that Sqd colocalizes with RbfN in Drosophila salivary gland cells. We also show that double RNAi knockdown of Rbf and Sqd in the eye results in an extensive loss of eye bristles, suggesting that Rbf and Sqd function in a common pathway. We conclude from our studies that Rbf physically and genetically interacts with Sqd. We propose that the retinoblastoma tumor suppressor may play a novel role in RNA processing through interaction with RNA-binding proteins.     

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.