Dyrk1a is required for craniofacial development in Xenopus laevis.

Loss of function variations in the dual specificity tyrosine-phosphorylation-regulated kinase 1 A (DYRK1A) gene are associated with craniofacial malformations in humans. Here we characterized the effects of deficient DYRK1A in craniofacial development using a developmental model, Xenopus laevis. Dyrk1a mRNA and protein were expressed throughout the developing head and both were enriched in the branchial arches which contribute to the face and jaw. Consistently, reduced Dyrk1a function, using dyrk1a morpholinos and pharmacological inhibitors, resulted in orofacial malformations including hypotelorism, altered mouth shape, slanted eyes, and narrower face accompanied by smaller jaw cartilage and muscle. Inhibition of Dyrk1a function resulted in misexpression of key craniofacial regulators including transcription factors and members of the retinoic acid signaling pathway. Two such regulators, sox9 and pax3 are required for neural crest development and their decreased expression corresponds with smaller neural crest domains within the branchial arches. Finally, we determined that the smaller size of the faces, jaw elements and neural crest domains in embryos deficient in Dyrk1a could be explained by increased cell death and decreased proliferation. This study is the first to provide insight into why craniofacial birth defects might arise in humans with variants of DYRK1A.

Proteasome inhibition paradoxically degrades gain-of-function mutant p53 R273H in NSCLC and could have therapeutic implications.

Lung cancer is the leading cause of cancer mortality. Despite therapeutic advances in recent years, new treatment strategies are needed to improve outcomes of lung cancer patients. Mutant p53 is prevalent in lung cancers and drives several hallmarks of cancer through a gain-of-function oncogenic program, and often predicts a poorer prognosis. The oncogenicity of mutant p53 is related to its stability and accumulation in cells by evading degradation by the proteasome. Therefore, destabilization of mutant p53 has been sought as a therapeutic strategy, but so far without clinical success. In this study, we report that proteasome inhibition results in degradation of mutant p53 in non-small cell lung cancer (NSCLC) cell lines bearing the R273H mutant protein and show evidence that this was mediated by hsp70. NSCLC cell lines with the mutant R273H allele demonstrated increased susceptibility and apoptosis to proteasome inhibitors. These data suggest that proteasome inhibitors could have therapeutic implications in some subsets of TP53 mutated NSCLC.

Dyrk1a is required for craniofacial development in Xenopus laevis.

Loss of function mutations in the dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) gene are associated with craniofacial malformations in humans. Here we characterized the effects of deficient DYRK1A in craniofacial development using a developmental model, Xenopus laevis . Dyrk1a mRNA and protein was expressed throughout the developing head and was enriched in the branchial arches which contribute to the face and jaw. Consistently, reduced Dyrk1a function, using dyrk1a morpholinos and pharmacological inhibitors, resulted in orofacial malformations including hypotelorism, altered mouth shape, slanted eyes, and narrower face accompanied by smaller jaw cartilage and muscle. Inhibition of Dyrk1a function resulted in misexpression of key craniofacial regulators including transcription factors and members of the retinoic acid signaling pathway. Two such regulators, sox9 and pax3 are required for neural crest development and their decreased expression corresponds with smaller neural crest domains within the branchial arches. Finally, we determined that the smaller size of the faces, jaw elements and neural crest domains in embryos deficient in Dyrk1a could be explained by increased cell death and decreased proliferation. This study is the first to provide insight into why craniofacial birth defects might arise in humans with DYRK1A mutations.

Insights from the protein interaction Universe of the multifunctional "Goldilocks" kinase DYRK1A.

Human Dual specificity tyrosine (Y)-Regulated Kinase 1A (DYRK1A) is encoded by a dosage-dependent gene located in the Down syndrome critical region of human chromosome 21. The known substrates of DYRK1A include proteins involved in transcription, cell cycle control, DNA repair and other processes. However, the function and regulation of this kinase is not fully understood, and the current knowledge does not fully explain the dosage-dependent function of this kinase. Several recent proteomic studies identified DYRK1A interacting proteins in several human cell lines. Interestingly, several of known protein substrates of DYRK1A were undetectable in these studies, likely due to a transient nature of the kinase-substrate interaction. It is possible that the stronger-binding DYRK1A interacting proteins, many of which are poorly characterized, are involved in regulatory functions by recruiting DYRK1A to the specific subcellular compartments or distinct signaling pathways. Better understanding of these DYRK1A-interacting proteins could help to decode the cellular processes regulated by this important protein kinase during embryonic development and in the adult organism. Here, we review the current knowledge of the biochemical and functional characterization of the DYRK1A protein-protein interaction network and discuss its involvement in human disease.

Exploitation of Sulfated Glycosaminoglycan Status for Precision Medicine of Triplatin in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer lacking targetable biomarkers. TNBC is known to be most aggressive and when metastatic is often drug-resistant and uncurable. Biomarkers predicting response to therapy improve treatment decisions and allow personalized approaches for patients with TNBC. This study explores sulfated glycosaminoglycan (sGAG) levels as a predictor of TNBC response to platinum therapy. sGAG levels were quantified in three distinct TNBC tumor models, including cell line-derived, patient-derived xenograft (PDX) tumors, and isogenic models deficient in sGAG biosynthesis. The in vivo antitumor efficacy of Triplatin, a sGAG-directed platinum agent, was compared in these models with the clinical platinum agent, carboplatin. We determined that >40% of TNBC PDX tissue microarray samples have high levels of sGAGs. The in vivo accumulation of Triplatin in tumors as well as antitumor efficacy of Triplatin positively correlated with sGAG levels on tumor cells, whereas carboplatin followed the opposite trend. In carboplatin-resistant tumor models expressing high levels of sGAGs, Triplatin decreased primary tumor growth, reduced lung metastases, and inhibited metastatic growth in lungs, liver, and ovaries. sGAG levels served as a predictor of Triplatin sensitivity in TNBC. Triplatin may be particularly beneficial in treating patients with chemotherapy-resistant tumors who have evidence of residual disease after standard neoadjuvant chemotherapy. More effective neoadjuvant and adjuvant treatment will likely improve clinical outcome of TNBC.

Simultaneous expression of MMB-FOXM1 complex components enables efficient bypass of senescence.

Cellular senescence is a stable cell cycle arrest that normal cells undergo after a finite number of divisions, in response to a variety of intrinsic and extrinsic stimuli. Although senescence is largely established and maintained by the p53/p21WAF1/CIP1 and pRB/p16INK4A tumour suppressor pathways, the downstream targets responsible for the stability of the growth arrest are not known. We have employed a stable senescence bypass assay in conditionally immortalised human breast fibroblasts (CL3EcoR) to investigate the role of the DREAM complex and its associated components in senescence. DREAM is a multi-subunit complex comprised of the MuvB core, containing LIN9, LIN37, LIN52, LIN54, and RBBP4, that when bound to p130, an RB1 like protein, and E2F4 inhibits cell cycle-dependent gene expression thereby arresting cell division. Phosphorylation of LIN52 at Serine 28 is required for DREAM assembly. Re-entry into the cell cycle upon phosphorylation of p130 leads to disruption of the DREAM complex and the MuvB core, associating initially to B-MYB and later to FOXM1 to form MMB and MMB-FOXM1 complexes respectively. Here we report that simultaneous expression of MMB-FOXM1 complex components efficiently bypasses senescence with LIN52, B-MYB, and FOXM1 as the crucial components. Moreover, bypass of senescence requires non-phosphorylated LIN52 that disrupts the DREAM complex, thereby indicating a central role for assembly of the DREAM complex in senescence.

DREAM On: Cell Cycle Control in Development and Disease.

Perfectly orchestrated periodic gene expression during cell cycle progression is essential for maintaining genome integrity and ensuring that cell proliferation can be stopped by environmental signals. Genetic and proteomic studies during the past two decades revealed remarkable evolutionary conservation of the key mechanisms that control cell cycle-regulated gene expression, including multisubunit DNA-binding DREAM complexes. DREAM complexes containing a retinoblastoma family member, an E2F transcription factor and its dimerization partner, and five proteins related to products of Caenorhabditis elegans multivulva (Muv) class B genes lin-9, lin-37, lin-52, lin-53, and lin-54 (comprising the MuvB core) have been described in diverse organisms, from worms to humans. This review summarizes the current knowledge of the structure, function, and regulation of DREAM complexes in different organisms, as well as the role of DREAM in human disease.

Oncogenic B-Myb Is Associated With Deregulation of the DREAM-Mediated Cell Cycle Gene Expression Program in High Grade Serous Ovarian Carcinoma Clinical Tumor Samples.

Cell cycle control drives cancer progression and treatment response in high grade serous ovarian carcinoma (HGSOC). MYBL2 (encoding B-Myb), an oncogene with prognostic significance in several cancers, is highly expressed in most HGSOC cases; however, the clinical significance of B-Myb in this disease has not been well-characterized. B-Myb is associated with cell proliferation through formation of the MMB (Myb and MuvB core) protein complex required for transcription of mitotic genes. High B-Myb expression disrupts the formation of another transcriptional cell cycle regulatory complex involving the MuvB core, DREAM (DP, RB-like, E2F, and MuvB), in human cell lines. DREAM coordinates cell cycle dependent gene expression by repressing over 800 cell cycle genes in G0/G1. Here, we take a bioinformatics approach to further evaluate the effect of B-Myb expression on DREAM target genes in HGSOC and validate our cellular model with clinical specimens. We show that MYBL2 is highly expressed in HGSOC and correlates with expression of DREAM and MMB target genes in both The Cancer Genome Atlas (TCGA) as well as independent analyses of HGSOC primary tumors (N = 52). High B-Myb expression was also associated with poor overall survival in the TCGA cohort and analysis by a DREAM target gene expression signature yielded a negative impact on survival. Together, our data support the conclusion that high expression of MYBL2 is associated with deregulation of DREAM/MMB-mediated cell cycle gene expression programs in HGSOC and may serve as a prognostic factor independent of its cell cycle role. This provides rationale for further, larger scale studies aimed to determine the clinical predictive value of the B-Myb gene expression signature for treatment response as well as patient outcomes.

Progesterone Receptors Promote Quiescence and Ovarian Cancer Cell Phenotypes via DREAM in p53-Mutant Fallopian Tube Models.

CONTEXT: The ability of ovarian steroids to modify ovarian cancer (OC) risk remains controversial. Progesterone is considered to be protective; recent studies indicate no effect or enhanced OC risk. Knowledge of progesterone receptor (PR) signaling during altered physiology that typifies OC development is limited. OBJECTIVE: This study defines PR-driven oncogenic signaling mechanisms in p53-mutant human fallopian tube epithelia (hFTE), a precursor of the most aggressive OC subtype. METHODS: PR expression in clinical samples of serous tubal intraepithelial carcinoma (STIC) lesions and high-grade serous OC (HGSC) tumors was analyzed. Novel PR-A and PR-B isoform-expressing hFTE models were characterized for gene expression and cell cycle progression, emboli formation, and invasion. PR regulation of the DREAM quiescence complex and DYRK1 kinases was established. RESULTS: STICs and HGSC express abundant activated phospho-PR. Progestin promoted reversible hFTE cell cycle arrest, spheroid formation, and invasion. RNAseq/biochemical studies revealed potent ligand-independent/-dependent PR actions, progestin-induced regulation of the DREAM quiescence complex, and cell cycle target genes through enhanced complex formation and chromatin recruitment. Disruption of DREAM/DYRK1s by pharmacological inhibition, HPV E6/E7 expression, or DYRK1A/B depletion blocked progestin-induced cell arrest and attenuated PR-driven gene expression and associated OC phenotypes. CONCLUSION: Activated PRs support quiescence and pro-survival/pro-dissemination cell behaviors that may contribute to early HGSC progression. Our data support an alternative perspective on the tenet that progesterone always confers protection against OC. STICs can reside undetected for decades prior to invasive disease; our studies reveal clinical opportunities to prevent the ultimate development of HGSC by targeting PRs, DREAM, and/or DYRKs.

PAF remodels the DREAM complex to bypass cell quiescence and promote lung tumorigenesis.

The DREAM complex orchestrates cell quiescence and the cell cycle. However, how the DREAM complex is deregulated in cancer remains elusive. Here, we report that PAF (PCLAF/KIAA0101) drives cell quiescence exit to promote lung tumorigenesis by remodeling the DREAM complex. PAF is highly expressed in lung adenocarcinoma (LUAD) and is associated with poor prognosis. Importantly, Paf knockout markedly suppressed LUAD development in mouse models. PAF depletion induced LUAD cell quiescence and growth arrest. PAF is required for the global expression of cell-cycle genes controlled by the repressive DREAM complex. Mechanistically, PAF inhibits DREAM complex formation by binding to RBBP4, a core DREAM subunit, leading to transactivation of DREAM target genes. Furthermore, pharmacological mimicking of PAF-depleted transcriptomes inhibited LUAD tumor growth. Our results unveil how the PAF-remodeled DREAM complex bypasses cell quiescence to promote lung tumorigenesis and suggest that the PAF-DREAM axis may be a therapeutic vulnerability in lung cancer.

Restoring the DREAM Complex Inhibits the Proliferation of High-Risk HPV Positive Human Cells.

High-risk (HR) human papillomaviruses are known causative agents in 5% of human cancers including cervical, ano-genital and head and neck carcinomas. In part, HR-HPV causes cancer by targeting host-cell tumor suppressors including retinoblastoma protein (pRb) and RB-like proteins p107 and p130. HR-HPV E7 uses a LxCxE motif to bind RB proteins, impairing their ability to control cell-cycle dependent transcription. E7 disrupts DREAM (Dimerization partner, RB-like, E2F and MuvB), a transcriptional repressor complex that can include p130 or p107, but not pRb, which regulates genes required for cell cycle progression. However, it is not known whether disruption of DREAM plays a significant role in HPV-driven tumorigenesis. In the DREAM complex, LIN52 is an adaptor that binds directly to p130 via an E7-like LxSxE motif. Replacement of the LxSxE sequence in LIN52 with LxCxE (LIN52-S20C) increases p130 binding and partially restores DREAM assembly in HPV-positive keratinocytes and human cervical cancer cells, inhibiting proliferation. Our findings demonstrate that disruption of the DREAM complex by E7 is an important process promoting cellular proliferation by HR-HPV. Restoration of the DREAM complex in HR-HPV positive cells may therefore have therapeutic benefits in HR-HPV positive cancers.

Preparing Immunoprecipitations for Immunoblotting.

Immunoprecipitated proteins can be readily analyzed by immunoblotting. Proteins can be efficiently eluted from the Protein A or similar beads by addition of the SDS-PAGE sample loading buffer and heating at 95°C. This elution procedure will also remove the capturing antibody from the beads unless the antibody was cross-linked to the beads. Alternatively, the immunoprecipitated proteins as well as non-cross-linked capture antibodies can be eluted from the beads using low (2.1-2.8) or high (10-11) pH conditions. Incubation of the immunoprecipitates with the excess of the competing peptide allows the elution of the captured proteins without contamination of the sample with the antibodies present in the immunoprecipitates. However, this option is not always available, and the cost of competing peptide can be prohibitive for the routine immunoprecipitation/immunoblotting experiments. In this protocol, elution of the immunoprecipitated proteins from the beads is performed by mixing Protein A or similar beads containing the immunoprecipitated protein antigens of interest with SDS-PAGE sample buffer and boiling to prepare samples for protein gel electrophoresis.

Blocking of Immunoblots and Incubation with Antibodies: Procedure for Immunoblots Prepared with Immunoprecipitated Protein Antigens.

Detection of the protein antigens in immunoblots prepared with immunoprecipitated protein antigens can be affected by the presence of high amounts of the immunoprecipitating antibodies. When it is not possible to use the immunoprecipitating antibodies and the primary antibodies raised in different species, this protocol provides a convenient and inexpensive alternative to achieve optimal detection of immunoprecipitated protein antigens. In this protocol, a nitrocellulose or polyvinylidene fluoride membrane containing immunoprecipitated protein samples is rinsed with ultrapure H2O after the transfer of proteins and detection of the total proteins using Ponceau S dye (optional). Blocking solution is applied to the membrane, and the membrane is incubated and then rinsed off (optional) before addition of the primary antibody labeled with biotin. After washing, the membrane is incubated with enzyme- or fluorochrome-labeled avidin for detection.

Immunoblotting.

Immunoblotting allows detection of a protein antigen immobilized on the protein-retaining membrane support such as nitrocellulose or polyvinylidene fluoride (PVDF). The detection of the protein of interest relies on the binding of an antibody that specifically recognizes the protein of interest exposed on the membrane. The protein of interest can be purified or mixed with other proteins as in cell or tissue extracts. Usually immunoblotting combines the resolution of proteins by gel electrophoresis with immunochemical detection and is referred to as "western blotting." Immunoblotting can be used to determine the presence and the steady-state level of the protein of interest in the sample, its relative molecular weight, and the distribution of the protein between cellular fractions. Immunoblotting can be performed using the antibodies raised against synthetic peptide antigens modified to mimic posttranslational modifications of proteins, such as phosphorylation and acetylation, to study these modifications in the protein of interest in vivo. When antibodies against the protein of interest are not available, immunoblotting can be performed using antibodies that specifically recognize the recombinant epitope tags (hemagglutinin [HA]-, Flag-, cMyc-, or glutathione-S-transferase [GST]) fused to the protein of interest using recombinant DNA techniques. Immunoblotting has a variety of research, clinical, and forensic medicine applications. It is also one of the standard techniques for characterization of antibodies from different samples of polyclonal sera or hybridoma supernatants.

CtBP determines ovarian cancer cell fate through repression of death receptors.

C-terminal binding protein 2 (CtBP2) is elevated in epithelial ovarian cancer, especially in the aggressive and highly lethal subtype, high-grade serous ovarian cancer (HGSOC). However, whether HGSOC tumor progression is dependent on CtBP2 or its paralog CtBP1, is not well understood. Here we report that CtBP1/2 repress HGSOC cell apoptosis through silencing of death receptors (DRs) 4/5. CtBP1 or 2 knockdown upregulated DR4/5 expression, and triggered autonomous apoptosis via caspase 8 activation, but dependent on cell-type context. Activation of DR4/5 by CtBP1/2 loss also sensitized HGSOC cell susceptibility to the proapoptotic DR4/5 ligand TRAIL. Consistent with its function as transcription corepressor, CtBP1/2 bound to the promoter regions of DR4/5 and repressed DR4/5 expression, presumably through recruitment to a repressive transcription regulatory complex. We also found that CtBP1 and 2 were both required for repression of DR4/5. Collectively, this study identifies CtBP1 and 2 as potent repressors of DR4/5 expression and activity, and supports the targeting of CtBP as a promising therapeutic strategy for HGSOC.

Staining the Blot for Total Protein with Ponceau S.

Before probing blots for the presence of an antigen, the total composition of the transferred proteins can be determined by staining the nitrocellulose or polyvinylidene fluoride (PVDF) membrane. Staining for proteins is useful to determine the position of the non-prestained molecular weight markers or individual lanes on the gel and to ensure that efficient transfer has occurred. It can be also used to verify equal loading of the samples in the gel when a comparison of the protein of interest between the different samples is important. The conventional procedures such as Coomassie Blue and silver staining methods used for staining polyacrylamide gels are incompatible with immunoblotting. Ponceau S is the more common staining method in immunoblotting protocols because it is compatible with antibody-antigen binding, is cost efficient, and provides a good contrast between the stained bands and background. In this protocol, nitrocellulose or PVDF membrane is rinsed with ultrapure H2O after the transfer of proteins. Ponceau S dye is applied as an acidic aqueous solution, and the proteins on the membrane are stained with red color. The membrane is briefly destained with water and can be photographed or scanned to obtain the image of the total protein staining. Individual lane positions or the molecular weight standards can be marked with a pencil, if required.

Stripping of the Immunoblot for Reprobing.

For most immunoblots developed with chemiluminescence or with fluorochrome-based detection systems, it is possible to remove the primary and secondary antibodies from the membrane without affecting the bound antigen. This allows you to reuse the membrane for detection of another protein antigen. The blots developed with chromogenic substrates can also be stripped of antibodies and reprobed, but the bands detected in the first round of immunoblotting will remain unaffected. Stripping and reprobing of the membrane are particularly useful when the amount of sample is limited or when it is important to accurately compare the signal between two different protein antigens in the same sample. Examples of such experiments include determining the levels of a protein antigen in a series of samples relative to the loading control and comparison of the phosphorylated form to the total levels of the protein in the sample.

Blocking of Immunoblots and Incubation with Antibodies: Procedure for Immunoblots Prepared with Whole-Cell Lysates or Purified Proteins.

In this protocol, immunoblots are prepared for detection of the proteins of interest by incubation with blocking solution to prevent nonspecific antibody binding, followed by incubation with primary and secondary antibodies. When labeled primary antibody is used, incubation with secondary antibodies can be omitted.

Detection of an Antigen on an Immunoblot.

Detection of the antigen on an immunoblot can be achieved by either enzyme-based detection systems or using detection reagents labeled with fluorochromes. Two major types of enzyme-based detection systems are used in immunoblotting based on either horseradish peroxidase (HRP)-or alkaline phosphatase (AP)-coupled antibodies, and a range of soluble substrates that yield insoluble colored products (chromogenic detection) or generate light (chemiluminescent detection). This protocol describes chromogenic detection with AP as well as both chromogenic and chemiluminescent methods of detection with HRP. Before the detection step, the nitrocellulose or polyvinylidene fluoride (PVDF) membrane is incubated with the antigen using enzyme-conjugated primary antibodies or secondary detection reagents (such as species-specific anti-immunoglobulin antibodies, streptavidin, or Protein A) and washed. An appropriate substrate solution is applied to the membrane, and the signal is detected in the course of the enzymatic reaction, resulting either in development of insoluble colored products (chromogenic substrates) or in generation of light (chemiluminescent substrates). This protocol also describes use of detection reagents labeled with fluorochromes. The nitrocellulose or PVDF membrane incubated with fluorochrome-labeled detection reagents is rinsed with PBS and processed for image capturing with appropriate imaging equipment.

Freezing Cell Pellets for Large-Scale Immunoprecipitation.

The advance, large-scale preparation of 108-109 adherent or suspension cells before the performance of immunoprecipitation can be advantageous given the time commitment required. The freezing of cells before lysis can preserve protein-protein interactions and posttranslational modifications that may otherwise become denatured and/or degraded upon initiation of cell lysis. This method can also be applied to the preparation of adherent or suspension cells on a smaller scale and is especially useful when multiple time points are being investigated over the course of several days or weeks. Cells are grown under optimal culturing conditions to promote a high degree of viability before being rinsed twice in phosphate-buffered saline (PBS), scraped into a polypropylene tube, and pelleted by centrifugation. The resulting cell pellet is frozen and can be stored for several months at -80°C.