Placental co-transcriptional activator Vestigial-like 1 (VGLL1) drives tumorigenesis via increasing transcription of proliferation and invasion genes.

Introduction: Vestigial-like 1 (VGLL1) is a co-transcriptional activator that binds to TEA domain-containing transcription factors (TEADs). Its expression is upregulated in a variety of aggressive cancer types, including pancreatic and basal-like breast cancer, and increased transcription of VGLL1 is strongly correlated with poor prognosis and decreased overall patient survival. In normal tissues, VGLL1 is most highly expressed within placental trophoblast cells, which share the common attributes of rapid cellular proliferation and invasion with tumor cells. The impact of VGLL1 in cancer has not been fully elucidated and no VGLL1-targeted therapy currently exists. Methods: The aim of this study was to evaluate the cellular function and downstream genomic targets of VGLL1 in placental, pancreatic, and breast cancer cells. Functional assays were employed to assess the role of VGLL1 in cellular invasion and proliferation, and ChIP-seq and RNAseq assays were performed to identify VGLL1 target genes and potential impact using pathway analysis. Results: ChIP-seq analysis identified eight transcription factors with a VGLL1-binding motif that were common between all three cell types, including TEAD1-4, AP-1, and GATA6, and revealed ~3,000 shared genes with which VGLL1 interacts. Furthermore, increased VGLL1 expression led to an enhancement of cell invasion and proliferation, which was supported by RNAseq analysis showing transcriptional changes in several genes known to be involved in these processes. Discussion: This work expands our mechanistic understanding of VGLL1 function in tumor cells and provides a strong rationale for developing VGLL1-targeted therapies for treating cancer patients.

Epidermal Growth Factor Receptor-Targeted Neoantigen Peptide Vaccination for the Treatment of Non-Small Cell Lung Cancer and Glioblastoma.

The epidermal growth factor receptor (EGFR) plays crucial roles in several important biological functions such as embryogenesis, epithelial tissue development, and cellular regeneration. However, in multiple solid tumor types overexpression and/or activating mutations of the EGFR gene frequently occur, thus hijacking the EGFR signaling pathway to promote tumorigenesis. Non-small cell lung cancer (NSCLC) tumors in particular often contain prevalent and shared EGFR mutations that provide an ideal source for public neoantigens (NeoAg). Studies in both humans and animal models have confirmed the immunogenicity of some of these NeoAg peptides, suggesting that they may constitute viable targets for cancer immunotherapies. Peptide vaccines targeting mutated EGFR have been tested in multiple clinical trials, demonstrating an excellent safety profile and encouraging clinical efficacy. For example, the CDX-110 (rindopepimut) NeoAg peptide vaccine derived from the EGFRvIII deletion mutant in combination with temozolomide and radiotherapy has shown efficacy in treating EGFRvIII-harboring glioblastoma multiforme (GBM) patients undergone surgery in multiple Phase I and II clinical trials. Furthermore, pilot clinical trials that have administered personalized NeoAg peptides for treating advanced-stage NSCLC patients have shown this approach to be a feasible and safe method to increase antitumor immune responses. Amongst the vaccine peptides administered, EGFR mutation-targeting NeoAgs induced the strongest T cell-mediated immune responses in patients and were also associated with objective clinical responses, implying a promising future for NeoAg peptide vaccines for treating NSCLC patients with selected EGFR mutations. The efficacy of NeoAg-targeting peptide vaccines may be further improved by combining with other modalities such as tyrosine kinase or immune checkpoint inhibitor (ICI) therapy, which are currently being tested in animal models and clinical trials. Herein, we review the most current basic and clinical research progress on EGFR-targeted peptide vaccination for the treatment of NSCLC and other solid tumor types.

Charge-based interactions through peptide position 4 drive diversity of antigen presentation by human leukocyte antigen class I molecules.

Human leukocyte antigen class I (HLA-I) molecules bind and present peptides at the cell surface to facilitate the induction of appropriate CD8+ T cell-mediated immune responses to pathogen- and self-derived proteins. The HLA-I peptide-binding cleft contains dominant anchor sites in the B and F pockets that interact primarily with amino acids at peptide position 2 and the C-terminus, respectively. Nonpocket peptide-HLA interactions also contribute to peptide binding and stability, but these secondary interactions are thought to be unique to individual HLA allotypes or to specific peptide antigens. Here, we show that two positively charged residues located near the top of peptide-binding cleft facilitate interactions with negatively charged residues at position 4 of presented peptides, which occur at elevated frequencies across most HLA-I allotypes. Loss of these interactions was shown to impair HLA-I/peptide binding and complex stability, as demonstrated by both in vitro and in silico experiments. Furthermore, mutation of these Arginine-65 (R65) and/or Lysine-66 (K66) residues in HLA-A*02:01 and A*24:02 significantly reduced HLA-I cell surface expression while also reducing the diversity of the presented peptide repertoire by up to 5-fold. The impact of the R65 mutation demonstrates that nonpocket HLA-I/peptide interactions can constitute anchor motifs that exert an unexpectedly broad influence on HLA-I-mediated antigen presentation. These findings provide fundamental insights into peptide antigen binding that could broadly inform epitope discovery in the context of viral vaccine development and cancer immunotherapy.

Evolution of CD8+ T Cell Receptor (TCR) Engineered Therapies for the Treatment of Cancer.

Engineered T cell receptor T (TCR-T) cell therapy has facilitated the generation of increasingly reliable tumor antigen-specific adaptable cellular products for the treatment of human cancer. TCR-T cell therapies were initially focused on targeting shared tumor-associated peptide targets, including melanoma differentiation and cancer-testis antigens. With recent technological developments, it has become feasible to target neoantigens derived from tumor somatic mutations, which represents a highly personalized therapy, since most neoantigens are patient-specific and are rarely shared between patients. TCR-T therapies have been tested for clinical efficacy in treating solid tumors in many preclinical studies and clinical trials all over the world. However, the efficacy of TCR-T therapy for the treatment of solid tumors has been limited by a number of factors, including low TCR avidity, off-target toxicities, and target antigen loss leading to tumor escape. In this review, we discuss the process of deriving tumor antigen-specific TCRs, including the identification of appropriate tumor antigen targets, expansion of antigen-specific T cells, and TCR cloning and validation, including techniques and tools for TCR-T cell vector construction and expression. We highlight the achievements of recent clinical trials of engineered TCR-T cell therapies and discuss the current challenges and potential solutions for improving their safety and efficacy, insights that may help guide future TCR-T studies in cancer.

Neoantigen vaccination induces clinical and immunologic responses in non-small cell lung cancer patients harboring EGFR mutations.

BACKGROUND: Neoantigen (NeoAg) peptides displayed at the tumor cell surface by human leukocyte antigen molecules show exquisite tumor specificity and can elicit T cell mediated tumor rejection. However, few NeoAgs are predicted to be shared between patients, and none to date have demonstrated therapeutic value in the context of vaccination. METHODS: We report here a phase I trial of personalized NeoAg peptide vaccination (PPV) of 24 stage III/IV non-small cell lung cancer (NSCLC) patients who had previously progressed following multiple conventional therapies, including surgery, radiation, chemotherapy, and tyrosine kinase inhibitors (TKIs). Primary endpoints of the trial evaluated feasibility, tolerability, and safety of the personalized vaccination approach, and secondary trial endpoints assessed tumor-specific immune reactivity and clinical responses. Of the 16 patients with epidermal growth factor receptor (EGFR) mutations, nine continued TKI therapy concurrent with PPV and seven patients received PPV alone. RESULTS: Out of 29 patients enrolled in the trial, 24 were immunized with personalized NeoAg peptides. Aside from transient rash, fatigue and/or fever observed in three patients, no other treatment-related adverse events were observed. Median progression-free survival and overall survival of the 24 vaccinated patients were 6.0 and 8.9 months, respectively. Within 3-4 months following initiation of PPV, seven RECIST-based objective clinical responses including one complete response were observed. Notably, all seven clinical responders had EGFR-mutated tumors, including four patients that had continued TKI therapy concurrently with PPV. Immune monitoring showed that five of the seven responding patients demonstrated vaccine-induced T cell responses against EGFR NeoAg peptides. Furthermore, two highly shared EGFR mutations (L858R and T790M) were shown to be immunogenic in four of the responding patients, all of whom demonstrated increases in peripheral blood neoantigen-specific CD8+ T cell frequencies during the course of PPV. CONCLUSIONS: These results show that personalized NeoAg vaccination is feasible and safe for advanced-stage NSCLC patients. The clinical and immune responses observed following PPV suggest that EGFR mutations constitute shared, immunogenic neoantigens with promising immunotherapeutic potential for large subsets of NSCLC patients. Furthermore, PPV with concurrent EGFR inhibitor therapy was well tolerated and may have contributed to the induction of PPV-induced T cell responses.

Vestigial-like 1 is a shared targetable cancer-placenta antigen expressed by pancreatic and basal-like breast cancers.

Cytotoxic T lymphocyte (CTL)-based cancer immunotherapies have shown great promise for inducing clinical regressions by targeting tumor-associated antigens (TAA). To expand the TAA landscape of pancreatic ductal adenocarcinoma (PDAC), we performed tandem mass spectrometry analysis of HLA class I-bound peptides from 35 PDAC patient tumors. This identified a shared HLA-A*0101 restricted peptide derived from co-transcriptional activator Vestigial-like 1 (VGLL1) as a putative TAA demonstrating overexpression in multiple tumor types and low or absent expression in essential normal tissues. Here we show that VGLL1-specific CTLs expanded from the blood of a PDAC patient could recognize and kill in an antigen-specific manner a majority of HLA-A*0101 allogeneic tumor cell lines derived not only from PDAC, but also bladder, ovarian, gastric, lung, and basal-like breast cancers. Gene expression profiling reveals VGLL1 as a member of a unique group of cancer-placenta antigens (CPA) that may constitute immunotherapeutic targets for patients with multiple cancer types.

RNA editing derived epitopes function as cancer antigens to elicit immune responses.

In addition to genomic mutations, RNA editing is another major mechanism creating sequence variations in proteins by introducing nucleotide changes in mRNA sequences. Deregulated RNA editing contributes to different types of human diseases, including cancers. Here we report that peptides generated as a consequence of RNA editing are indeed naturally presented by human leukocyte antigen (HLA) molecules. We provide evidence that effector CD8+ T cells specific for edited peptides derived from cyclin I are present in human tumours and attack tumour cells that are presenting these epitopes. We show that subpopulations of cancer patients have increased peptide levels and that levels of edited RNA correlate with peptide copy numbers. These findings demonstrate that RNA editing extends the classes of HLA presented self-antigens and that these antigens can be recognised by the immune system.

The RNA-binding Protein MEX3B Mediates Resistance to Cancer Immunotherapy by Downregulating HLA-A Expression.

Purpose: Cancer immunotherapy has shown promising clinical outcomes in many patients. However, some patients still fail to respond, and new strategies are needed to overcome resistance. The purpose of this study was to identify novel genes and understand the mechanisms that confer resistance to cancer immunotherapy.Experimental Design: To identify genes mediating resistance to T-cell killing, we performed an open reading frame (ORF) screen of a kinome library to study whether overexpression of a gene in patient-derived melanoma cells could inhibit their susceptibility to killing by autologous tumor-infiltrating lymphocytes (TIL).Results: The RNA-binding protein MEX3B was identified as a top candidate that decreased the susceptibility of melanoma cells to killing by TILs. Further analyses of anti-PD-1-treated melanoma patient tumor samples suggested that higher MEX3B expression is associated with resistance to PD-1 blockade. In addition, significantly decreased levels of IFNγ were secreted from TILs incubated with MEX3B-overexpressing tumor cells. Interestingly, this phenotype was rescued upon overexpression of exogenous HLA-A2. Consistent with this, we observed decreased HLA-A expression in MEX3B-overexpressing tumor cells. Finally, luciferase reporter assays and RNA-binding protein immunoprecipitation assays suggest that this is due to MEX3B binding to the 3' untranslated region (UTR) of HLA-A to destabilize the mRNA.Conclusions: MEX3B mediates resistance to cancer immunotherapy by binding to the 3' UTR of HLA-A to destabilize the HLA-A mRNA and thus downregulate HLA-A expression on the surface of tumor cells, thereby making the tumor cells unable to be recognized and killed by T cells. Clin Cancer Res; 24(14); 3366-76. ©2018 AACRSee related commentary by Kalbasi and Ribas, p. 3239.

HSP90 inhibition enhances cancer immunotherapy by upregulating interferon response genes.

T-cell-based immunotherapies are promising treatments for cancer patients. Although durable responses can be achieved in some patients, many patients fail to respond to these therapies, underscoring the need for improvement with combination therapies. From a screen of 850 bioactive compounds, we identify HSP90 inhibitors as candidates for combination with immunotherapy. We show that inhibition of HSP90 with ganetespib enhances T-cell-mediated killing of patient-derived human melanoma cells by their autologous T cells in vitro and potentiates responses to anti-CTLA4 and anti-PD1 therapy in vivo. Mechanistic studies reveal that HSP90 inhibition results in upregulation of interferon response genes, which are essential for the enhanced killing of ganetespib treated melanoma cells by T cells. Taken together, these findings provide evidence that HSP90 inhibition can potentiate T-cell-mediated anti-tumor immune responses, and rationale to explore the combination of immunotherapy and HSP90 inhibitors.Many patients fail to respond to T cell based immunotherapies. Here, the authors, through a high-throughput screening, identify HSP90 inhibitors as a class of preferred drugs for treatment combination with immunotherapy.

SLC45A2: A Melanoma Antigen with High Tumor Selectivity and Reduced Potential for Autoimmune Toxicity.

Cytotoxic T lymphocyte (CTL)-based immunotherapies have had remarkable success at generating objective clinical responses in patients with advanced metastatic melanoma. Although the melanocyte differentiation antigens (MDA) MART-1, PMEL, and tyrosinase were among the first melanoma tumor-associated antigens identified and targeted with immunotherapy, expression within normal melanocytes of the eye and inner ear can elicit serious autoimmune side effects, thus limiting their clinical potential as CTL targets. Using a tandem mass spectrometry (MS) approach to analyze the immunopeptidomes of 55 melanoma patient-derived cell lines, we identified a number of shared HLA class I-bound peptides derived from the melanocyte-specific transporter protein SLC45A2. Antigen-specific CTLs generated against HLA-A*0201- and HLA-A*2402-restricted SLC45A2 peptides effectively killed a majority of HLA-matched cutaneous, uveal, and mucosal melanoma cell lines tested (18/25). CTLs specific for SLC45A2 showed significantly reduced recognition of HLA-matched primary melanocytes that were, conversely, robustly killed by MART1- and PMEL-specific T cells. Transcriptome analysis revealed that SLC45A2 mRNA expression in normal melanocytes was less than 2% that of other MDAs, therefore providing a more favorable melanoma-to-melanocyte expression ratio. Expression of SLC45A2 and CTL sensitivity could be further upregulated in BRAF(V600E)-mutant melanoma cells upon treatment with BRAF or MEK inhibitors, similarly to other MDAs. Taken together, our study demonstrates the feasibility of using tandem MS as a means of discovering shared immunogenic tumor-associated epitopes and identifies SLC45A2 as a promising immunotherapeutic target for melanoma with high tumor selectivity and reduced potential for autoimmune toxicity. Cancer Immunol Res; 5(8); 618-29. ©2017 AACR.

Trouble at the core: BRAF(V600E) drives multiple modes of T-cell suppression in melanoma.

Several studies have demonstrated that oncogenic BRAF(V600E) promotes T-cell suppression in melanoma by upregulating the transcription of a multitude of immunomodulatory chemokine and cytokine genes. BRAF(V600E) has now been shown to act even more directly to evade cytotoxic T-cell recognition, by driving rapid internalization of human leukocyte antigen (HLA) class I from the tumor-cell surface and its intracellular sequestration.

Grancalcin (GCA) modulates Toll-like receptor 9 (TLR9) mediated signaling through its direct interaction with TLR9.

Toll-like receptors (TLRs) are playing important roles in stimulating the innate immune response and intensifying adaptive immune response against invading pathogens. Appropriate regulation of TLR activation is important to maintain a balance between preventing tumor activation and inhibiting autoimmunity. Toll-like receptor 9 (TLR9) senses microbial DNA in the endosomes of plasmacytoid dendritic cells and triggers myeloid differentiation primary response gene 88 (MyD88) dependent nuclear factor kappa B (NF-κB) pathways and type I interferon (IFN) responses. However, mechanisms of how TLR9 signals are mediated and which molecules are involved in controlling TLR9 functions remain poorly understood. Here, we report that penta EF-hand protein grancalcin (GCA) interacts and binds with TLR9 in a yeast two-hybrid system and an overexpression system. Using siRNA-mediated knockdown experiments, we also revealed that GCA positively regulates type I IFN production, cytokine/chemokine production through nuclear localization of interferon regulatory factor 7 (IRF7), NF-κB activation, and mitogen-activated protein kinase (MAPK) activation in plasmacytoid dendritic cells. Our results indicate that heterodimerization of GCA and TLR9 is important for TLR9-mediated downstream signaling and might serve to fine tune processes against viral infection.

BRAFV600E Co-opts a Conserved MHC Class I Internalization Pathway to Diminish Antigen Presentation and CD8+ T-cell Recognition of Melanoma.

Oncogene activation in tumor cells induces broad and complex cellular changes that contribute significantly to disease initiation and progression. In melanoma, oncogenic BRAF(V600E) has been shown to drive the transcription of a specific gene signature that can promote multiple mechanisms of immune suppression within the tumor microenvironment. We show here that BRAF(V600E) also induces rapid internalization of MHC class I (MHC-I) from the melanoma cell surface and its intracellular sequestration within endolysosomal compartments. Importantly, MAPK inhibitor treatment quickly restored MHC-I surface expression in tumor cells, thereby enhancing melanoma antigen-specific T-cell recognition and effector function. MAPK pathway-driven relocalization of HLA-A*0201 required a highly conserved cytoplasmic serine phosphorylation site previously implicated in rapid MHC-I internalization and recycling by activated immune cells. Collectively, these data suggest that oncogenic activation of BRAF allows tumor cells to co-opt an evolutionarily conserved MHC-I trafficking pathway as a strategy to facilitate immune evasion. This link between MAPK pathway activation and the MHC-I cytoplasmic tail has direct implications for immunologic recognition of tumor cells and provides further evidence to support testing therapeutic strategies combining MAPK pathway inhibition with immunotherapies in the clinical setting.

Phospholipid scramblase 1 regulates Toll-like receptor 9-mediated type I interferon production in plasmacytoid dendritic cells.

Toll-like receptor 9 (TLR9) senses microbial DNA in the endosomes of plasmacytoid dendritic cells (pDCs) and triggers MyD88-dependent type I interferon (IFN) responses. To better understand TLR9 biology in pDCs, we established a yeast two-hybrid library for the identification of TLR9-interacting proteins. Here, we report that an IFN-inducible protein, phospholipid scramblase 1 (PLSCR1), interacts with TLR9 in pDCs. Knockdown of PLSCR1 expression by siRNA in human pDC cell line led to a 60-70% reduction of IFN-α responses following CpG-ODN (oligodeoxynucleotide) stimulation. Primary pDCs from PLSCR1-deficient mice produced lower amount of type 1 IFN than pDCs from the wild-type mice in response to CpG-ODN, herpes simplex virus and influenza A virus. Following CpG-A stimulation, there were much lower amounts of TLR9 in the early endosomes together with CpG-A in pDCs from PLSCR1-deficient mice. Our study demonstrates that PLSCR1 is a TLR9-interacting protein that plays an important role in pDC's type 1 IFN responses by regulating TLR9 trafficking to the endosomal compartment.

Metastasis-associated protein 1 transgenic mice: a new model of spontaneous B-cell lymphomas.

Metastasis-associated protein 1 (MTA1), a component of the nuclear remodeling complex and the founding homologue of the MTA family, has been implicated in metastasis, but definitive causative evidence in an animal model system is currently lacking. Here, we show that MTA1 overexpression in transgenic mice is accompanied by a high incidence of spontaneous B cell lymphomas including diffuse large B cell lymphomas (DLBCL). Lymphocytes and lymphoma cells from MTA1-TG mice are hyperproliferative. Lymphomas were transplantable and of clonal origin and were characterized by down-regulation of p27Kip1 as well as up-regulation of Bcl2 and cyclin D1. The significance of these murine studies was established by evidence showing a widespread up-regulation of MTA1 in DLBCL from humans. These findings reveal a previously unrecognized role for the MTA1 pathway in the development of spontaneous B cell lymphomas, and offer a potential therapeutic target in B cell lymphomas. These observations suggest that MTA1-TG mice represent a new model of spontaneous DLBCL associated with high tumor incidence and could be used for therapeutic intervention studies.

Identification of Pax5 as a target of MTA1 in B-cell lymphomas.

Previously, we have shown that metastasis-associated protein 1 (MTA1) overexpression in transgenic mice was accompanied by high incidence of spontaneous B-cell lymphomas including diffuse large B-cell lymphomas (DLBCL). To understand the molecular basis of lymphoma in MTA1-transgenic (MTA1-TG) mice, we wished to identify a putative MTA1 target with a causal role in B-cell lymphogenesis. Using chromatin immunoprecipitation assays, we identified paired box gene 5 (Pax5), a molecule previously implicated in B-cell lymphogenesis, as a potential downstream effector of MTA1. Lymphomas from MTA1-TG mice also showed up-regulation of Pax5. We also found that MTA1 acetylated on Lys(626) interacted with p300 histone acetyltransferase, and that acetylated MTA1 was recruited to the Pax5 promoter to stimulate Pax5 transcription. Global gene profiling identified down-regulation of a set of genes, including those downstream of Pax5 and directly implicated in the B-cell lymphogenesis. Significance of these murine studies was established by evidence showing a widespread up-regulation of both MTA1 and Pax5 in DLBCL from humans. These observations provide in vivo genetic evidence for a role of MTA1 in lymphomagenesis.

Repression of Six3 by a corepressor regulates rhodopsin expression.

Here, we provide gain-of-function, loss-of function, and molecular evidence supporting genetic interactions between metastasis associated protein 1 (MTA1) and Six3 and between Six3 and rhodopsin. We discovered that MTA1 physically interacts with the Six3 chromatin in a histone deacetylase-dependent manner, leading to transcriptional suppression of the Six3 gene. MTA1 is also a Six3-interacting corepressor that contributes to a self-negative regulation of Six3 transcription by Six3. In contrast, deletion of the MTA1 alleles in murine embryonic fibroblasts or its knockdown in rat retinal ganglion cells stimulates Six3 expression. MTA1 inactivation in the MTA1-null mice results in an elevated Six3 level and proliferation of the retina cells with no obvious abnormities in eye formation. However, unexpectedly, we discovered an enhanced recruitment of Six3 to the rhodopsin chromatin in retina from the MTA1-null mice; Six3's homeodomain interacts with specific DNA elements in the rhodopsin promoter to stimulate its transcription, resulting in increased rhodopsin expression. Further, in holoprosencephaly patients, Six3 protein with a naturally occurring deletion mutation in the helix 3 of the homeodomain does not bind to rhodopsin DNA or stimulate rhodopsin transcription, implying a potential defective rhodopsin pathway in the affected holoprosencephaly patients. Further Six3 cooperates with Crx or NRL in stimulating transcription from the rhodopsin-luc. These findings reveal a previously unrecognized role for the MTA1 as an upstream modifier of Six3 and indicate that Six3 is a direct stimulator of rhodopsin expression, thus revealing a putative role for the MTA1/Six3/rhodopsin pathway in vertebrate eye.

Phosphorylation-dependent regulation of nuclear localization and functions of integrin-linked kinase.

Integrin-linked kinase (ILK) is a phosphorylated protein that regulates physiological processes that overlap with those regulated by p21-activated kinase 1 (PAK1). Here we report the possible role of ILK phosphorylation by PAK1 in ILK-mediated signaling and intracellular translocation. We found that PAK1 phosphorylates ILK at threonine-173 and serine-246 in vitro and in vivo. Depletion of PAK1 decreased the levels of endogenous ILK phosphorylation in vivo. Mutation of PAK1 phosphorylation sites on ILK to alanine reduced cell motility and cell proliferation. Biochemical fractionation, confocal microscopy, and chromatin-interaction analyses of human cells revealed that ILK localizes predominantly in the cytoplasm but also resides in the nucleus. Transfection of MCF-7 cells with point mutants ILK-T173A, ILK-S246A, or ILK-T173A; S246A (ILK-DM) altered ILK localization. Selective depletion of PAK1 dramatically increased the nuclear and focal point accumulation of ILK, further demonstrating a role for PAK1 in ILK translocation. We also identified functional nuclear localization sequence and nuclear export sequence motifs in ILK, delineated an apparently integral role for ILK in maintaining normal nuclear integrity, and established that ILK interacts with the regulatory region of the CNKSR3 gene chromatin to negatively modulate its expression. Together, these results suggest that ILK is a PAK1 substrate, undergoes phosphorylation-dependent shuttling between the cell nucleus and cytoplasm, and interacts with gene-regulatory chromatin.

Signaling-dependent and coordinated regulation of transcription, splicing, and translation resides in a single coregulator, PCBP1.

Transcription, splicing, and translation are potentially coordinately regulatable in a temporospatial-dependent manner, although supporting experimental evidence for this notion is scarce. Yeast two-hybrid screening of a mammary gland cDNA library with human p21-activated kinase 1 (Pak1) as bait identified polyC-RNA-binding protein 1 (PCBP1), which controls translation from mRNAs containing the DICE (differentiation control element). Mitogenic stimulation of human cells phosphorylated PCBP1 on threonines 60 and 127 in a Pak1-sensitive manner. Pak1-dependent phosphorylation of PCBP1 released its binding and translational inhibition from a DICE-minigene. Overexpression of PCBP1 also inhibited the translation of the endogenous L1 cell adhesion molecule mRNA, which contains two DICE motifs in the 3' untranslated region. We also found that Pak1 activation led to an increased nuclear retention of PCBP1, recruitment to the eukaryotic translation initiation factor 4E (eIF4E) promoter, and stimulation of eIF4E expression in a Pak1-sensitive manner. Moreover, mitogenic stimulation promoted Pak1- and PCBP1-dependent alternative splicing and exon inclusion from a CD44 minigene. The alternative splicing functions of PCBP1 were in turn mediated by its intrinsic interaction with Caper alpha, a U2 snRNP auxiliary factor-related protein previously implicated in RNA splicing. These findings establish the principle that a single coregulator can function as a signal-dependent and coordinated regulator of transcription, splicing, and translation.