Sustained antigen delivery improves germinal center reaction and increases antibody responses in neonatal mice.

Neonates and young infants are known to have limited responses to pediatric vaccines due to reduced germinal center formation. Extended vaccine antigen dosing was previously shown to expand germinal center formation and improve humoral responses in adult mice. We report that sustained antigen delivery through sequential dosing overcomes neonatal limitations to form germinal center reactions and improves humoral immunity. Thus, vaccine strategies that extend the release of vaccine antigens may reduce the number of doses, and time needed, to achieve protective immunity in neonates and young infants.

IL6 suppresses vaccine responses in neonates by enhancing IL2 activity on T follicular helper cells.

The inability of neonates to develop CD4+FoxP3-CXCR5hiPD-1hi T follicular helper (TFH) cells contributes to their weak vaccine responses. In previous studies, we measured diminished IgG responses when IL-6 was co-injected with a pneumococcal conjugate vaccine (PCV) in neonatal mice. This is in sharp contrast to adults, where IL-6 improves vaccine responses by downregulating the expression of IL-2Rß on TFH cells and protecting them from the inhibitory effect of IL-2. In this study, we found that splenic IL-6 levels rapidly increased in both adult and neonatal mice following immunization, but the increase in neonatal mice was significantly more than that of adult mice. Moreover, immunized neonatal TFH cells expressed significantly more IL-2 as well as its receptors, IL-2Rα and IL-2Rß, than the adult cells. Remarkably, IL-6 co-injection with PCV vaccine further increased the production of IL-2 and the expression of its receptors by neonatal TFH cells, whereas excess IL-6 had totally opposite effect in immunized adult mice. Underscoring the role of IL-6 in activating the IL-2 mediated suppression of vaccine responses, immunization of IL-6 knock-out neonates led to improved antibody responses accompanied by expanded TFH cells as well as lower levels of IL-2 and IL-2 receptors on TFH cells. Moreover, CpG containing PCV improved TFH response in neonates by suppressing the expression of IL-2 receptors on TFH cells and inhibiting IL-2 activity. These findings unveil age-specific differences in IL-6 mediated vaccine responses and highlight the need to consider age-related immunobiological attributes in designing vaccines.

Scaffold coupling: ERK activation by trans-phosphorylation across different scaffold protein species.

RAS-ERK (extracellular signal-regulated kinase) pathway signals are modulated by scaffold proteins that assemble the components of different kinase tiers into a sequential phosphorylation cascade. In the prevailing model scaffold proteins function as isolated entities, where the flux of phosphorylation events progresses downstream linearly, to achieve ERK phosphorylation. We show that different types of scaffold proteins, specifically KSR1 (kinase suppressor of Ras 1) and IQGAP1 (IQ motif-containing guanosine triphosphatase activating protein 1), can bind to each other, forming a complex whereby phosphorylation reactions occur across both species. MEK (mitogen-activated protein kinase kinase) bound to IQGAP1 can phosphorylate ERK docked at KSR1, a process that we have named "trans-phosphorylation." We also reveal that ERK trans-phosphorylation participates in KSR1-regulated adipogenesis, and it also underlies the modest cytotoxicity exhibited by KSR-directed inhibitors. Overall, we identify interactions between scaffold proteins and trans-phosphorylation as an additional level of regulation in the ERK cascade, with broad implications in signaling and the design of scaffold protein-aimed therapeutics.

Calmodulin influences MAPK signaling by binding KSR1.

The mitogen-activated protein kinase (MAPK) cascade is a fundamental signaling pathway that regulates cell fate decisions in response to external stimuli. Several scaffold proteins bind directly to kinase components of this pathway and regulate their activation by growth factors. One of the best studied MAPK scaffolds is kinase suppressor of Ras1 (KSR1), which is induced by epidermal growth factor (EGF) to translocate to the plasma membrane where it activates extracellular signal-regulated kinase (ERK). While Ca2+ has been shown to modulate MAPK signaling, the molecular mechanisms by which this occurs are incompletely understood. Here we tested the hypothesis that Ca2+ alters MAPK activity at least in part via KSR1. Using several approaches, including fusion proteins, immunoprecipitation, confocal microscopy, and a cell-permeable chemical inhibitor, we investigated the functional interaction between KSR1 and calmodulin. In vitro analysis with pure proteins reveals that calmodulin binds directly to KSR1. Moreover, endogenous calmodulin and KSR1 co-immunoprecipitate from mammalian cell lysates. Importantly, Ca2+ is required for the association between calmodulin and KSR1, both in vitro and in cells. The cell-permeable calmodulin antagonist CGS9343B significantly reduced activation of ERK by EGF in mouse embryo fibroblasts that overexpress KSR1, but not in control cells. Moreover, CGS9343B impaired the ability of EGF to induce KSR1 translocation to the plasma membrane and to stimulate formation of KSR1-ERK and KSR1-pERK (phosphorylated ERK) complexes in cells. Collectively, our data identify a previously unrecognized mechanism by which the scaffold protein KSR1 couples Ca2+ and calmodulin signaling to the MAPK cascade.

Genome-Wide Analysis Unveils DNA Helicase RECQ1 as a Regulator of Estrogen Response Pathway in Breast Cancer Cells.

Susceptibility to breast cancer is significantly increased in individuals with germ line mutations in RECQ1 (also known as RECQL or RECQL1), a gene encoding a DNA helicase essential for genome maintenance. We previously reported that RECQ1 expression predicts clinical outcomes for sporadic breast cancer patients stratified by estrogen receptor (ER) status. Here, we utilized an unbiased integrative genomics approach to delineate a cross talk between RECQ1 and ERα, a known master regulatory transcription factor in breast cancer. We found that expression of ESR1, the gene encoding ERα, is directly activated by RECQ1. More than 35% of RECQ1 binding sites were cobound by ERα genome-wide. Mechanistically, RECQ1 cooperates with FOXA1, the pioneer transcription factor for ERα, to enhance chromatin accessibility at the ESR1 regulatory regions in a helicase activity-dependent manner. In clinical ERα-positive breast cancers treated with endocrine therapy, high RECQ1 and high FOXA1 coexpressing tumors were associated with better survival. Collectively, these results identify RECQ1 as a novel cofactor for ERα and uncover a previously unknown mechanism by which RECQ1 regulates disease-driving gene expression in ER-positive breast cancer cells.

IQGAP1 binds AMPK and is required for maximum AMPK activation.

AMP-activated protein kinase (AMPK) is a fundamental component of a protein kinase cascade that is an energy sensor. AMPK maintains energy homeostasis in the cell by promoting catabolic and inhibiting anabolic pathways. Activation of AMPK requires phosphorylation by the liver kinase B1 or by the Ca2+/calmodulin-dependent protein kinase 2 (CaMKK2). The scaffold protein IQGAP1 regulates intracellular signaling pathways, such as the mitogen-activated protein kinase and AKT signaling cascades. Recent work implicates the participation of IQGAP1 in metabolic function, but the molecular mechanisms underlying these effects are poorly understood. Here, using several approaches including binding analysis with fusion proteins, siRNA-mediated gene silencing, RT-PCR, and knockout mice, we investigated whether IQGAP1 modulates AMPK signaling. In vitro analysis reveals that IQGAP1 binds directly to the α1 subunit of AMPK. In addition, we observed a direct interaction between IQGAP1 and CaMKK2, which is mediated by the IQ domain of IQGAP1. Both CaMKK2 and AMPK associate with IQGAP1 in cells. The ability of metformin and increased intracellular free Ca2+ concentrations to activate AMPK is reduced in cells lacking IQGAP1. Importantly, Ca2+-stimulated AMPK phosphorylation was rescued by re-expression of IQGAP1 in IQGAP1-null cell lines. Comparison of the fasting response in wild-type and IQGAP1-null mice revealed that transcriptional regulation of the gluconeogenesis genes PCK1 and G6PC and the fatty acid synthesis genes FASN and ACC1 is impaired in IQGAP1-null mice. Our data disclose a previously unidentified functional interaction between IQGAP1 and AMPK and suggest that IQGAP1 modulates AMPK signaling.

Keratin 19 regulates cell cycle pathway and sensitivity of breast cancer cells to CDK inhibitors.

Keratin 19 (K19) belongs to the keratin family of proteins, which maintains structural integrity of epithelia. In cancer, K19 is highly expressed in several types where it serves as a diagnostic marker. Despite the positive correlation between higher expression of K19 in tumor and worse patient survival, the role of K19 in breast cancer remains unclear. Therefore, we ablated K19 expression in MCF7 breast cancer cells and found that K19 was required for cell proliferation. Transcriptome analyses of KRT19 knockout cells identified defects in cell cycle progression and levels of target genes of E2F1, a key transcriptional factor for the transition into S phase. Furthermore, proper levels of cyclin dependent kinases (CDKs) and cyclins, including D-type cyclins critical for E2F1 activation, were dependent on K19 expression, and K19-cyclin D co-expression was observed in human breast cancer tissues. Importantly, K19 interacts with cyclin D3, and a loss of K19 resulted in decreased protein stability of cyclin D3 and sensitivity of cells towards CDK inhibitor-induced cell death. Overall, these findings reveal a novel function of K19 in the regulation of cell cycle program and suggest that K19 may be used to predict the efficacy of CDK inhibitors for treatments of breast cancer.

The DNA repair helicase RECQ1 has a checkpoint-dependent role in mediating DNA damage responses induced by gemcitabine.

The response of cancer cells to therapeutic drugs that cause DNA damage depends on genes playing a role in DNA repair. RecQ-like helicase 1 (RECQ1), a DNA repair helicase, is critical for genome stability, and loss-of-function mutations in the RECQ1 gene are associated with increased susceptibility to breast cancer. In this study, using a CRISPR/Cas9-edited cell-based model, we show that the genetic or functional loss of RECQ1 sensitizes MDA-MB-231 breast cancer cells to gemcitabine, a nucleoside analog used in chemotherapy for triple-negative breast cancer. RECQ1 loss led to defective ATR Ser/Thr kinase (ATR)/checkpoint kinase 1 (ChK1) activation and greater DNA damage accumulation in response to gemcitabine treatment. Dual deficiency of MUS81 structure-specific endonuclease subunit (MUS81) and RECQ1 increased gemcitabine-induced, replication-associated DNA double-stranded breaks. Consistent with defective checkpoint activation, a ChK1 inhibitor further sensitized RECQ1-deficient cells to gemcitabine and increased cell death. Our results reveal an important role for RECQ1 in controlling cell cycle checkpoint activation in response to gemcitabine-induced replication stress.

RECQ1 expression is upregulated in response to DNA damage and in a p53-dependent manner.

Sensitivity of cancer cells to DNA damaging chemotherapeutics is determined by DNA repair processes. Consequently, cancer cells may upregulate the expression of certain DNA repair genes as a mechanism to promote chemoresistance. Here, we report that RECQ1, a breast cancer susceptibility gene that encodes the most abundant RecQ helicase in humans, is a p53-regulated gene, potentially acting as a defense against DNA damaging agents. We show that RECQ1 mRNA and protein levels are upregulated upon treatment of cancer cells with a variety of DNA damaging agents including the DNA-alkylating agent methylmethanesulfonate (MMS). The MMS-induced upregulation of RECQ1 expression is p53-dependent as it was observed in p53-proficient but not in isogenic p53-deficient cells. The RECQ1 promoter is bound by endogenous p53 and is responsive to p53 in luciferase reporter assays suggesting that RECQ1 is a direct target of p53. Treatment with the chemotherapeutic drugs temozolomide and fotemustine also increased RECQ1 mRNA levels whereas depletion of RECQ1 enhanced cellular sensitivity to these agents. These results identify a previously unrecognized p53-mediated upregulation of RECQ1 expression in response to DNA damage and implicate RECQ1 in the repair of DNA lesions including those induced by alkylating and other chemotherapeutic agents.

Long Noncoding RNA PURPL Suppresses Basal p53 Levels and Promotes Tumorigenicity in Colorectal Cancer.

Basal p53 levels are tightly suppressed under normal conditions. Disrupting this regulation results in elevated p53 levels to induce cell cycle arrest, apoptosis, and tumor suppression. Here, we report the suppression of basal p53 levels by a nuclear, p53-regulated long noncoding RNA that we termed PURPL (p53 upregulated regulator of p53 levels). Targeted depletion of PURPL in colorectal cancer cells results in elevated basal p53 levels and induces growth defects in cell culture and in mouse xenografts. PURPL associates with MYBBP1A, a protein that binds to and stabilizes p53, and inhibits the formation of the p53-MYBBP1A complex. In the absence of PURPL, MYBBP1A interacts with and stabilizes p53. Silencing MYBBP1A significantly rescues basal p53 levels and proliferation in PURPL-deficient cells, suggesting that MYBBP1A mediates the effect of PURPL in regulating p53. These results reveal a p53-PURPL auto-regulatory feedback loop and demonstrate a role for PURPL in maintaining basal p53 levels.

A new sub-pathway of long-patch base excision repair involving 5' gap formation.

Base excision repair (BER) is one of the most frequently used cellular DNA repair mechanisms and modulates many human pathophysiological conditions related to DNA damage. Through live cell and in vitro reconstitution experiments, we have discovered a major sub-pathway of conventional long-patch BER that involves formation of a 9-nucleotide gap 5' to the lesion. This new sub-pathway is mediated by RECQ1 DNA helicase and ERCC1-XPF endonuclease in cooperation with PARP1 poly(ADP-ribose) polymerase and RPA The novel gap formation step is employed during repair of a variety of DNA lesions, including oxidative and alkylation damage. Moreover, RECQ1 regulates PARP1 auto-(ADP-ribosyl)ation and the choice between long-patch and single-nucleotide BER, thereby modulating cellular sensitivity to DNA damage. Based on these results, we propose a revised model of long-patch BER and a new key regulation point for pathway choice in BER.

Clinicopathological and Functional Significance of RECQL1 Helicase in Sporadic Breast Cancers.

RECQL1, a key member of the RecQ family of DNA helicases, is required for DNA replication and DNA repair. Two recent studies have shown that germline RECQL1 mutations are associated with increased breast cancer susceptibility. Whether altered RECQL1 expression has clinicopathologic significance in sporadic breast cancers is unknown. We evaluated RECQL1 at the transcriptomic level (METABRIC cohort, n = 1,977) and at the protein level [cohort 1, n = 897; cohort 2, n = 252; cohort 3 (BRCA germline deficient), n = 74]. In RECQL1-depleted breast cancer cells, we investigated anthracycline sensitivity. High RECQL1 mRNA was associated with intClust.3 (P = 0.026), which is characterized by low genomic instability. On the other hand, low RECQL1 mRNA was linked to intClust.8 [luminal A estrogen receptor-positive (ER+) subgroup; P = 0.0455] and intClust.9 (luminal B ER+ subgroup; P = 0.0346) molecular phenotypes. Low RECQL1 expression was associated with shorter breast cancer-specific survival (P = 0.001). At the protein level, low nuclear RECQL1 level was associated with larger tumor size, lymph node positivity, high tumor grade, high mitotic index, pleomorphism, dedifferentiation, ER negativity, and HER-2 overexpression (P < 0.05). In ER+ tumors that received endocrine therapy, low RECQL1 was associated with poor survival (P = 0.008). However, in ER- tumors that received anthracycline-based chemotherapy, high RECQL1 was associated with poor survival (P = 0.048). In RECQL1-depleted breast cancer cell lines, we confirmed doxorubicin sensitivity, which was associated with DNA double-strand breaks accumulation, S-phase cell-cycle arrest, and apoptosis. We conclude that RECQL1 has prognostic and predictive significance in breast cancers. Mol Cancer Ther; 16(1); 239-50. ©2016 AACR.

Transcriptome guided identification of novel functions of RECQ1 helicase.

Gene expression changes in the functional absence of a specific RecQ protein, and how that relates to disease outcomes including cancer predisposition and premature aging in RecQ helicase associated syndromes, are poorly understood. Here we describe detailed experimental strategy for identification of RECQ1-regulated transcriptome that led us to uncover a novel association of RECQ1 in regulation of cancer cell migration and invasion. We initiated a focused study to determine whether RECQ1, the most abundant RecQ protein in humans, alters gene expression and also investigated whether RECQ1 binds with G4 motifs predicted to form G-quadruplex structures in the target gene promoters. Rescue of mRNA expression of select RECQ1-downregulated genes harboring G4 motifs required wild-type RECQ1 helicase. However, some RECQ1-regulated genes are also regulated by BLM and WRN proteins regardless of the presence or absence of G4 motifs. The approach described here is applicable for systematic comparison of gene expression signatures of individual RecQ proteins in isogenic background, and to elucidate their participation in transcription regulation through G-quadruplex recognition and/or resolution. Such strategies might also reveal molecular pathways that drive the pathogenesis of cancer and other diseases in specific RecQ deficiency.

RECQ1 interacts with FEN-1 and promotes binding of FEN-1 to telomeric chromatin.

RecQ helicases are a family of highly conserved proteins that maintain genomic stability through their important roles in replication restart mechanisms. Cellular phenotypes of RECQ1 deficiency are indicative of aberrant repair of stalled replication forks, but the molecular functions of RECQ1, the most abundant of the five known human RecQ homologues, have remained poorly understood. We show that RECQ1 associates with FEN-1 (flap endonuclease-1) in nuclear extracts and exhibits direct protein interaction in vitro. Recombinant RECQ1 significantly stimulated FEN-1 endonucleolytic cleavage of 5'-flap DNA substrates containing non-telomeric or telomeric repeat sequence. RECQ1 and FEN-1 were constitutively present at telomeres and their binding to the telomeric chromatin was enhanced following DNA damage. Telomere residence of FEN-1 was dependent on RECQ1 since depletion of RECQ1 reduced FEN-1 binding to telomeres in unperturbed cycling cells. Our results confirm a conserved collaboration of human RecQ helicases with FEN-1 and suggest both overlapping and specialized roles of RECQ1 in the processing of DNA structure intermediates proposed to arise during replication, repair and recombination.

Identification of RECQ1-regulated transcriptome uncovers a role of RECQ1 in regulation of cancer cell migration and invasion.

The RECQ protein family of helicases has critical roles in protecting and stabilizing the genome. Three of the 5 known members of the human RecQ family are genetically linked with cancer susceptibility syndromes, but the association of the most abundant human RecQ homolog, RECQ1, with cellular transformation is yet unclear. RECQ1 is overexpressed in a variety of human cancers, indicating oncogenic functions. Here, we assessed genome-wide changes in gene expression upon knockdown of RECQ1 in HeLa and MDA-MB-231 cells. Pathway analysis suggested that RECQ1 enhances the expression of multiple genes that play key roles in cell migration, invasion, and metastasis, including EZR, ITGA2, ITGA3, ITGB4, SMAD3, and TGFBR2. Consistent with these results, silencing RECQ1 significantly reduced cell migration and invasion. In comparison to genome-wide annotated promoter regions, the promoters of genes downregulated upon RECQ1 silencing were significantly enriched for a potential G4 DNA forming sequence motif. Chromatin immunoprecipitation assays demonstrated binding of RECQ1 to the G4 motifs in the promoters of select genes downregulated upon RECQ1 silencing. In breast cancer patients, the expression of a subset of RECQ1-activated genes positively correlated with RECQ1 expression. Moreover, high RECQ1 expression was associated with poor prognosis in breast cancer. Collectively, our findings identify a novel function of RECQ1 in gene regulation and indicate that RECQ1 contributes to tumor development and progression, in part, by regulating the expression of key genes that promote cancer cell migration, invasion and metastasis.

Human RECQ1 interacts with Ku70/80 and modulates DNA end-joining of double-strand breaks.

Genomic instability is a known precursor to cancer and aging. The RecQ helicases are a highly conserved family of DNA-unwinding enzymes that play key roles in maintaining genome stability in all living organisms. Human RecQ homologs include RECQ1, BLM, WRN, RECQ4, and RECQ5ß, three of which have been linked to diseases with elevated risk of cancer and growth defects (Bloom Syndrome and Rothmund-Thomson Syndrome) or premature aging (Werner Syndrome). RECQ1, the first RecQ helicase discovered and the most abundant in human cells, is the least well understood of the five human RecQ homologs. We have previously described that knockout of RECQ1 in mice or knockdown of its expression in human cells results in elevated frequency of spontaneous sister chromatid exchanges, chromosomal instability, increased load of DNA damage and heightened sensitivity to ionizing radiation. We have now obtained evidence implicating RECQ1 in the nonhomologous end-joining pathway of DNA double-strand break repair. We show that RECQ1 interacts directly with the Ku70/80 subunit of the DNA-PK complex, and depletion of RECQ1 results in reduced end-joining in cell free extracts. In vitro, RECQ1 binds and unwinds the Ku70/80-bound partial duplex DNA substrate efficiently. Linear DNA is co-bound by RECQ1 and Ku70/80, and DNA binding by Ku70/80 is modulated by RECQ1. Collectively, these results provide the first evidence for an interaction of RECQ1 with Ku70/80 and a role of the human RecQ helicase in double-strand break repair through nonhomologous end-joining.

Replication stress induces specific enrichment of RECQ1 at common fragile sites FRA3B and FRA16D.

BACKGROUND: Stalled replication forks at common fragile sites are a major cause of genomic instability. RecQ helicases, a highly conserved family of DNA-unwinding enzymes, are believed to ease 'roadblocks' that pose challenge to replication fork progression. Among the five known RecQ homologs in humans, functions of RECQ1, the most abundant of all, are poorly understood. We previously determined that RECQ1 helicase preferentially binds and unwinds substrates that mimic DNA replication/repair intermediates, and interacts with proteins involved in DNA replication restart mechanisms. METHOD: We have utilized chromatin immunoprecipitation followed by quantitative real-time PCR to investigate chromatin interactions of RECQ1 at defined genetic loci in the presence or absence of replication stress. We have also tested the sensitivity of RECQ1-depleted cells to aphidicolin induced replication stress. RESULTS: RECQ1 binds to the origins of replication in unperturbed cells. We now show that conditions of replication stress induce increased accumulation of RECQ1 at the lamin B2 origin in HeLa cells. Consistent with a role in promoting fork recovery or repair, RECQ1 is specifically enriched at two major fragile sites FRA3B and FRA16D where replication forks have stalled following aphidicolin treatment. RECQ1-depletion results in attenuated checkpoint activation in response to replication stress, increased sensitivity to aphidicolin and chromosomal instability. CONCLUSIONS: Given a recent biochemical observation that RECQ1 catalyzes strand exchange on stalled replication fork structures in vitro, our results indicate that RECQ1 facilitates repair of stalled or collapsed replication forks and preserves genome integrity. Our findings provide the first evidence of a crucial role for RECQ1 at naturally occurring fork stalling sites and implicate RECQ1 in mechanisms underlying common fragile site instability in cancer.