CDK8 and CDK19: positive regulators of signal-induced transcription and negative regulators of Mediator complex proteins.

We have conducted a detailed transcriptomic, proteomic and phosphoproteomic analysis of CDK8 and its paralog CDK19, alternative enzymatic components of the kinase module associated with transcriptional Mediator complex and implicated in development and diseases. This analysis was performed using genetic modifications of CDK8 and CDK19, selective CDK8/19 small molecule kinase inhibitors and a potent CDK8/19 PROTAC degrader. CDK8/19 inhibition in cells exposed to serum or to agonists of NFκB or protein kinase C (PKC) reduced the induction of signal-responsive genes, indicating a pleiotropic role of Mediator kinases in signal-induced transcriptional reprogramming. CDK8/19 inhibition under basal conditions initially downregulated a small group of genes, most of which were inducible by serum or PKC stimulation. Prolonged CDK8/19 inhibition or mutagenesis upregulated a larger gene set, along with a post-transcriptional increase in the proteins comprising the core Mediator complex and its kinase module. Regulation of both RNA and protein expression required CDK8/19 kinase activities but both enzymes protected their binding partner cyclin C from proteolytic degradation in a kinase-independent manner. Analysis of isogenic cell populations expressing CDK8, CDK19 or their kinase-inactive mutants revealed that CDK8 and CDK19 have the same qualitative effects on protein phosphorylation and gene expression at the RNA and protein levels, whereas differential effects of CDK8 versus CDK19 knockouts were attributable to quantitative differences in their expression and activity rather than different functions.

Suppression of HIV-TAT and cocaine-induced neurotoxicity and inflammation by cell penetrable itaconate esters.

HIV-associated neurological disorder (HAND) is a serious complication of HIV infection marked by neurotoxicity induced by viral proteins like Tat. Substance abuse exacerbates neurocognitive impairment in people living with HIV. There is an urgent need for therapeutic strategies to combat HAND comorbid with Cocaine Use Disorder (CUD). Our analysis of HIV and cocaine-induced transcriptomes in primary cortical cultures revealed significant overexpression of the macrophage-specific gene aconitate decarboxylase 1 (Acod1). The ACOD1 protein converts the tricarboxylic acid intermediate cis-aconitate into itaconate during the activation of inflammation. Itaconate then facilitates cytokine production and activates anti-inflammatory transcription factors, shielding macrophages from infection-induced cell death. However, the immunometabolic function of itaconate was unexplored in HIV and cocaine-exposed microglia. We assessed the potential of 4-octyl-itaconate (4OI), a cell-penetrable ester form of itaconate known for its anti-inflammatory properties. When primary cortical cultures exposed to Tat and cocaine were treated with 4OI, microglial cell number increased and the morphological altercations induced by Tat and cocaine were reversed. Microglial cells also appeared more ramified, resembling the quiescent microglia. 4OI treatment inhibited secretion of the proinflammatory cytokines IL-1α, IL-1ß, IL-6, and MIP1-α induced by Tat and cocaine. Transcriptome profiling determined that Nrf2 target genes were significantly activated in Tat and 4OI treated cultures relative to Tat alone. Further, genes associated with cytoskeleton dynamics in inflammatory microglia were downregulated by 4OI treatment. Together, the results strongly suggest 4-octyl-itaconate holds promise as a potential candidate for therapeutic development to treat HAND coupled with CUD comorbidities.

Glycosaminoglycan modifications of betaglycan regulate ectodomain shedding to fine-tune TGF-ß signaling responses in ovarian cancer.

In pathologies including cancer, aberrant Transforming Growth Factor-ß (TGF-ß) signaling exerts profound tumor intrinsic and extrinsic consequences. Intense clinical endeavors are underway to target this pathway. Central to the success of these interventions is pinpointing factors that decisively modulate the TGF-ß responses. Betaglycan/type III TGF-ß receptor (TßRIII), is an established co-receptor for the TGF-ß superfamily known to bind directly to TGF-ßs 1-3 and inhibin A/B. Betaglycan can be membrane-bound and also undergo ectodomain cleavage to produce soluble-betaglycan that can sequester its ligands. Its extracellular domain undergoes heparan sulfate and chondroitin sulfate glycosaminoglycan modifications, transforming betaglycan into a proteoglycan. We report the unexpected discovery that the heparan sulfate glycosaminoglycan chains on betaglycan are critical for the ectodomain shedding. In the absence of such glycosaminoglycan chains betaglycan is not shed, a feature indispensable for the ability of betaglycan to suppress TGF-ß signaling and the cells' responses to exogenous TGF-ß ligands. Using unbiased transcriptomics, we identified TIMP3 as a key inhibitor of betaglycan shedding thereby influencing TGF-ß signaling. Our results bear significant clinical relevance as modified betaglycan is present in the ascites of patients with ovarian cancer and can serve as a marker for predicting patient outcomes and TGF-ß signaling responses. These studies are the first to demonstrate a unique reliance on the glycosaminoglycan chains of betaglycan for shedding and influence on TGF-ß signaling responses. Dysregulated shedding of TGF-ß receptors plays a vital role in determining the response and availability of TGF-ßs', which is crucial for prognostic predictions and understanding of TGF-ß signaling dynamics.

Macrophage tumor necrosis factor-alpha deletion does not protect against obesity-associated metabolic dysfunction.

The pro-inflammatory cytokine, tumor necrosis factor-alpha (TNF-α), has been suggested to be a key factor in the induction of obesity-associated metabolic dysfunction. However, the role that macrophage-derived TNF-α has on regulating metabolic perturbations in obesity is not completely understood. Therefore, we utilized the TNF-αFlox/Flox(F/F) , LyzMcre± mouse model to determine the impact that macrophage TNF-α deletion has on the development of high-fat diet (HFD)-induced obesity. At 10 weeks of age, male littermates were randomly assigned to 1 of 4 groups: TNF-αF/F low-fat diet (TNF-αF/F LFD), TNF-αF/F,LyzMCre LFD, TNF-αF/F HFD, or TNF-αF/F,LyzMCre HFD (n = 16-28/group) and were fed their respective diets for 18 weeks. Body weight was assessed throughout the course of the experiment. Body composition, hepatic lipid accumulation, and metabolic outcomes were also examined. A microarray gene expression experiment was performed from RNA isolated from epididymal adipose tissue of the HFD-fed groups (n = 10/group) and results were verified via qRT-PCR for all groups. Macrophage-derived TNF-α deletion significantly reduced adipose tissue TNF-α gene expression and circulating TNF-α and downregulated genes linked to the toll-like receptor (TLR) and NFκB signaling pathways. However, macrophage TNF-α deletion had no effect on hindering the development of obesity, hepatic lipid accumulation, or improving glucose metabolism or insulin sensitivity. In conclusion, macrophage-derived TNF-α is not a causative factor for the induction of obesity-associated metabolic dysfunction.

Genomic variation in captive deer mouse (Peromyscus maniculatus) populations.

BACKGROUND: Deer mice (genus Peromyscus) are the most common rodents in North America. Despite the availability of reference genomes for some species, a comprehensive database of polymorphisms, especially in those maintained as living stocks and distributed to academic investigators, is missing. In the present study we surveyed two populations of P. maniculatus that are maintained at the Peromyscus Genetic Stock Center (PGSC) for polymorphisms across their 2.5 × 109 bp genome. RESULTS: High density of variation was identified, corresponding to one SNP every 55 bp for the high altitude stock (SM2) or 207 bp for the low altitude stock (BW) using snpEff (v4.3). Indels were detected every 1157 bp for BW or 311 bp for SM2. The average Watterson estimator for the BW and SM2 populations is 248813.70388 and 869071.7671 respectively. Some differences in the distribution of missense, nonsense and silent mutations were identified between the stocks, as well as polymorphisms in genes associated with inflammation (NFATC2), hypoxia (HIF1a) and cholesterol metabolism (INSIG1) and may possess value in modeling pathology. CONCLUSIONS: This genomic resource, in combination with the availability of P. maniculatus from the PGSC, is expected to promote genetic and genomic studies with this animal model.

PRDM1 silences stem cell-related genes and inhibits proliferation of human colon tumor organoids.

PRDM1 is a tumor suppressor that plays an important role in B and T cell lymphomas. Our previous studies demonstrated that PRDM1ß is a p53-response gene in human colorectal cancer cells. However, the function of PRDM1ß in colorectal cancer cells and colon tumor organoids is not clear. Here we show that PRDM1ß is a p53-response gene in human colon organoids and that low PRDM1 expression predicts poor survival in colon cancer patients. We engineered PRDM1 knockouts and overexpression clones in RKO cells and characterized the PRDM1-dependent transcript landscapes, revealing that both the α and ß transcript isoforms repress MYC-response genes and stem cell-related genes. Finally, we show that forced expression of PRDM1 in human colon cancer organoids prevents the formation and growth of colon tumor organoids in vitro. These results suggest that p53 may exert tumor-suppressive effects in part through a PRDM1-dependent silencing of stem cell genes, depleting the size of the normal intestinal stem cell compartment in response to DNA damage.

A strategy for the identification of paracrine regulators of cancer cell migration.

We hypothesized that the correlation of the whole transcriptome with quantifiable phenotypes may unveil genes contributing to the regulation of the corresponding response. We tested this hypothesis in cultured fibroblasts exposed to diverse pharmacological and biological agents, to identify genes influencing chemoattraction of breast cancer cells. Our analyses revealed several genes that correlated, either positively or negatively with cell migration, suggesting that they may operate as activators or inhibitors of this process. Survey of the scientific literature showed that genes exhibiting positive or negative association with cell migration had frequently been linked to cancer and metastasis before, while those with minimal association were not. The current methodology may formulate the basis for the development of novel strategies linking genes to quantifiable phenotypes.

Emodin Bidirectionally Modulates Macrophage Polarization and Epigenetically Regulates Macrophage Memory.

Macrophages are pleiotropic cells capable of performing a broad spectrum of functions. Macrophage phenotypes are classified along a continuum between the extremes of proinflammatory M1 macrophages and anti-inflammatory M2 macrophages. The seemingly opposing functions of M1 and M2 macrophages must be tightly regulated for an effective and proper response to foreign molecules or damaged tissue. Excessive activation of either M1 or M2 macrophages contributes to the pathology of many diseases. Emodin is a Chinese herb-derived compound and has shown potential to inhibit inflammation in various settings. In this study, we tested the ability of emodin to modulate the macrophage response to both M1 and M2 stimuli. Primary mouse macrophages were stimulated with LPS/IFNγ or IL4 with or without emodin, and the effects of emodin on gene transcription, cell signaling pathways, and histone modifications were examined by a variety of approaches, including microarray, quantitative real-time PCR, Western blotting, chromatin immunoprecipitation, and functional assays. We found that emodin bidirectionally tunes the induction of LPS/IFNγ- and IL4-responsive genes through inhibiting NFκB/IRF5/STAT1 signaling and IRF4/STAT6 signaling, respectively. Thereby, emodin modulates macrophage phagocytosis, migration, and NO production. Furthermore, emodin inhibited the removal of H3K27 trimethylation (H3K27m3) marks and the addition of H3K27 acetylation (H3K27ac) marks on genes required for M1 or M2 polarization of macrophages. In conclusion, our data suggest that emodin is uniquely able to suppress the excessive response of macrophages to both M1 and M2 stimuli and therefore has the potential to restore macrophage homeostasis in various pathologies.

IL-33 promotes growth and liver metastasis of colorectal cancer in mice by remodeling the tumor microenvironment and inducing angiogenesis.

Liver metastasis is the major cause of death from colorectal cancer (CRC). Understanding its mechanisms is necessary for timely diagnosis and development of effective therapies. Interleukin-33 (IL-33) is an IL-1 cytokine family member that uniquely functions as a cytokine and nuclear factor. It is released by necrotic epithelial cells and activated innate immune cells, functioning as an alarmin or an early danger signal. Its role in invoking type 2 immune response has been established; however, it has contrasting roles in tumor development and metastasis. We identified IL-33 as a potently upregulated cytokine in a highly metastatic murine CRC cell line and examined its role in tumor growth and metastasis to the liver. IL-33 was transgenically expressed in murine and human adenocarcinoma and carcinoma cell lines and their growth and spontaneous metastasis to the liver were assessed in orthotopic models of CRC in wild-type C57Bl/6 and Il33 knockout mice. The results showed that increased expression of IL-33 in CRC cells enhanced their tumor take, growth, and liver metastasis. Tumor- rather than host-derived IL-33 induced the enhanced recruitment of CD11b+ GR1+ and CD11b+ F4/80+ myeloid cells to remodel the tumor microenvironment by increased expression of mobilizing cytokines, and tumor angiogenesis by activating endothelial cells. IL-33 expression was elevated in patient tumor tissues, induced early in adenoma development, and activated by pro-inflammatory cytokines derived from the tumor microenvironment. The data suggest that tumor-derived IL-33 modulates the tumor microenvironment to potently promote colon carcinogenesis and liver metastasis, underscoring its potential as a therapeutic target. © 2016 Wiley Periodicals, Inc.

Prefrontal microRNA-221 Mediates Environmental Enrichment-Induced Increase of Locomotor Sensitivity to Nicotine.

BACKGROUND: Environmental enrichment alters susceptibility in developing drug addiction. We have demonstrated that rats raised in an enriched condition are more sensitive than rats raised in an impoverished condition to nicotine-induced locomotor activity, and this is associated with alterations of phosphorylated extracellular signal-regulated kinase 1/2 within the prefrontal cortex. This study determined the impact of microRNA-221 in the prefrontal cortex on phosphorylated extracellular signal-regulated kinase 1/2 and the enriched environment-dependent behavioral changes in response to nicotine. METHODS: A microRNA array was conducted to profile microRNA expression in the prefrontal cortex of enriched condition and impoverished condition rats in response to repeated nicotine (0.35 mg/kg, s.c.) administration. microRNA-221 in the prefrontal cortex, nucleus accumbens, and striatum was further verified by quantitative real-time PCR. Lentiviral-mediated overexpression of microRNA-221 in PC12 cells and the medial prefrontal cortex was performed to determine the effects of microRNA-221 on nicotine-mediated phosphorylated extracellular signal-regulated kinase 1/2, phosphorylated cAMP-response element-binding protein, and locomotor activity. RESULTS: microRNA-221 was profoundly upregulated in the prefrontal cortex but not in nucleus accumbens and striatum of enriched condition rats relative to impoverished condition rats following repeated administration of nicotine. Overexpression of lentiviral-microRNA-221 attenuated nicotine-induced increase in phosphorylated extracellular signal-regulated kinase 1/2 in PC12 cells. Lentiviral-microRNA-221 overexpression in the medial prefrontal cortex further increased locomotor activity in impoverished condition but not in enriched condition rats in response to repeated nicotine administration. Accordingly, lentiviral-microRNA-221 attenuated nicotine-induced increases in phosphorylated extracellular signal-regulated kinase 1/2 and phosphorylated cAMP-response element-binding protein in the medial prefrontal cortex of impoverished condition but not enriched condition rats. CONCLUSION: These findings suggest that environmental enrichment, via upregulation of prefrontal microRNA-221 expression, suppresses the nicotine-induced activation of extracellular signal-regulated kinase and cAMP-response element-binding protein, which provides a potential mechanism underlying enhanced locomotor sensitivity to nicotine.

Six1 promotes epithelial-mesenchymal transition and malignant conversion in human papillomavirus type 16-immortalized human keratinocytes.

Six1, a member of the Six family of homeodomain transcription factors, is overexpressed in various human cancers, and SIX1 overexpression is associated with tumor progression and metastasis. Six1 messenger RNA levels increase during in vitro progression of human papillomavirus type 16 (HPV16)-immortalized human keratinocytes (HKc/HPV16) toward a differentiation-resistant (HKc/DR) phenotype. In this study, we show that HKc/DR-overexpressing Six1 exhibited a more mesenchymal phenotype, as characterized by a fibroblastic appearance and increased invasion. We utilized Whole Human Genome Microarrays to explore the gene expression changes associated with Six1 overexpression in HKc/DR. We found that overexpression of Six1 downregulated epithelial-related genes and upregulated mesenchymal-related genes, which suggests that Six1 overexpression induces epithelial-mesenchymal transition (EMT). Pathway analysis of the microarray data showed alterations in the transforming growth factor-beta (TGF-ß) pathway, including enhanced expression of the TGF-ß receptor type II (TßRII), and activation of the mitogen-activated protein kinase (MAPK) pathway in HKc/DR-overexpressing Six1, suggesting that Smad-independent pathways of TGF-ß signaling may be involved in Six1-mediated EMT. p38 MAPK activation was required for sustained Six1-induced EMT and TßRII overexpression. Finally, we determined that Six1 overexpression in HKc/DR resulted in malignant conversion and increased the cancer stem cell (CSC)-like population. Thus, Six1 overexpression promotes EMT, CSCs properties and malignant conversion in HKc/DR through MAPK activation, which supports the possible use of p38-TßRII inhibitors for the treatment of cancers overexpressing Six1.

Identification of staphylococcal species based on variations in protein sequences (mass spectrometry) and DNA sequence (sodA microarray).

This report is among the first using sequence variation in newly discovered protein markers for staphylococcal (or indeed any other bacterial) speciation. Variation, at the DNA sequence level, in the sodA gene (commonly used for staphylococcal speciation) provided excellent correlation. Relatedness among strains was also assessed using protein profiling using microcapillary electrophoresis and pulsed field electrophoresis. A total of 64 strains were analyzed including reference strains representing the 11 staphylococcal species most commonly isolated from man (Staphylococcus aureus and 10 coagulase negative species [CoNS]). Matrix assisted time of flight ionization/ionization mass spectrometry (MALDI TOF MS) and liquid chromatography-electrospray ionization tandem mass spectrometry (LC ESI MS/MS) were used for peptide analysis of proteins isolated from gel bands. Comparison of experimental spectra of unknowns versus spectra of peptides derived from reference strains allowed bacterial identification after MALDI TOF MS analysis. After LC-MS/MS analysis of gel bands bacterial speciation was performed by comparing experimental spectra versus virtual spectra using the software X!Tandem. Finally LC-MS/MS was performed on whole proteomes and data analysis also employing X!tandem. Aconitate hydratase and oxoglutarate dehydrogenase served as marker proteins on focused analysis after gel separation. Alternatively on full proteomics analysis elongation factor Tu generally provided the highest confidence in staphylococcal speciation.

Reprint of "Identification of staphylococcal species based on variations in protein sequences (mass spectrometry) and DNA sequence (sodA microarray)".

This report is among the first using sequence variation in newly discovered protein markers for staphylococcal (or indeed any other bacterial) speciation. Variation, at the DNA sequence level, in the sodA gene (commonly used for staphylococcal speciation) provided excellent correlation. Relatedness among strains was also assessed using protein profiling using microcapillary electrophoresis and pulsed field electrophoresis. A total of 64 strains were analyzed including reference strains representing the 11 staphylococcal species most commonly isolated from man (Staphylococcus aureus and 10 coagulase negative species [CoNS]). Matrix assisted time of flight ionization/ionization mass spectrometry (MALDI TOF MS) and liquid chromatography-electrospray ionization tandem mass spectrometry (LC ESI MS/MS) were used for peptide analysis of proteins isolated from gel bands. Comparison of experimental spectra of unknowns versus spectra of peptides derived from reference strains allowed bacterial identification after MALDI TOF MS analysis. After LC-MS/MS analysis of gel bands bacterial speciation was performed by comparing experimental spectra versus virtual spectra using the software X!Tandem. Finally LC-MS/MS was performed on whole proteomes and data analysis also employing X!tandem. Aconitate hydratase and oxoglutarate dehydrogenase served as marker proteins on focused analysis after gel separation. Alternatively on full proteomics analysis elongation factor Tu generally provided the highest confidence in staphylococcal speciation.

Partial loss of Smad signaling during in vitro progression of HPV16-immortalized human keratinocytes.

BACKGROUND: Disruption of the transforming growth factor-beta (TGF-ß) signaling pathway is observed in many cancers, including cervical cancer, resulting in TGF-ß resistance. While normal human keratinocytes (HKc) and human papillomavirus type 16-immortalized HKc (HKc/HPV16) are sensitive to the growth inhibitory effects of TGF-ß, HKc/HPV16 develop resistance to TGF-ß1 as they progress in vitro to a differentiation resistant phenotype (HKc/DR). The loss of sensitivity to the antiproliferative effects of TGF-ß1 in HKc/DR is due, at least partially, to decreased expression of the TGF-ß receptor type I. In the present study, we explored in detail whether alterations in Smad protein levels, Smad phosphorylation, or nuclear localization of Smads in response to TGF-ß could contribute to the development of TGF-ß resistance during in vitro progression of HKc/HPV16, and whether TGF-ß induction of a Smad-responsive reporter gene was altered in HKc/DR. METHODS: Western blot analysis was used to assess Smad protein levels. In order to study Smad nuclear localization we performed indirect immunofluorescence. In addition, we determined Smad-mediated TGF-ß signaling using a luciferase reporter construct. RESULTS: We did not find a decrease in protein levels of Smad2, Smad3 or Smad4, or an increase in the inhibitory Smad7 that paralleled the loss of sensitivity to the growth inhibitory effects of TGF-ß1 observed in HKc/DR. However, we found diminished Smad2 phosphorylation, and delayed nuclear Smad3 localization in response to TGF-ß1 in HKc/DR, compared to normal HKc and TGF-ß sensitive HKc/HPV16. In addition, we determined that TGF-ß1 induction of a Smad responsive promoter is reduced by about 50% in HKc/DR, compared to HKc/HPV16. CONCLUSIONS: These results demonstrate that alterations in Smad protein levels are not associated with the loss of response to the antiproliferative effects of TGF-ß in HKc/DR, but that diminished and delayed Smad phosphorylation and nuclear localization, and decreased Smad signaling occur in response to TGF-ß in HKc/DR.

Suppression of HIV and cocaine-induced neurotoxicity and inflammation by cell penetrable itaconate esters.

HIV-associated neurological disorder (HAND) is a serious complication of HIV infection, marked by neurotoxicity induced by viral proteins like Tat. Substance abuse exacerbates neurocognitive impairment in people living with HIV. There is an urgent need for effective therapeutic strategies to combat HAND comorbid with Cocaine Use Disorder (CUD). Our analysis of the HIV and cocaine-induced transcriptomes in primary cortical cultures revealed a significant overexpression of the macrophage-specific gene, aconitate decarboxylase 1 (Acod1), caused by the combined insults of HIV and cocaine. ACOD1 protein converts the tricarboxylic acid intermediate cis-aconitate into itaconate during the activation of inflammation. The itaconate produced facilitates cytokine production and subsequently activates anti-inflammatory transcription factors, shielding macrophages from infection-induced cell death. While the role of itaconate' in limiting inflammation has been studied in peripheral macrophages, its immunometabolic function remains unexplored in HIV and cocaine-exposed microglia. We assessed in this model system the potential of 4-octyl-itaconate (4OI), a cell-penetrable esterified form of itaconate known for its potent anti-inflammatory properties and potential therapeutic applications. We administered 4OI to primary cortical cultures exposed to Tat and cocaine. 4OI treatment increased the number of microglial cells in both untreated and Tat±Cocaine-treated cultures and also reversed the morphological altercations induced by Tat and cocaine. In the presence of 4OI, microglial cells also appeared more ramified, resembling the quiescent microglia. Consistent with these results, 4OI treatment inhibited the secretion of the proinflammatory cytokines IL-1α, IL-1ß, IL-6, and MIP1-α induced by Tat and cocaine. Transcriptome profiling further determined that Nrf2 target genes such as NAD(P)H quinone oxidoreductase 1 (Nqo1), Glutathione S-transferase Pi (Gstp1), and glutamate cysteine ligase catalytic (Gclc), were most significantly activated in Tat-4OI treated cultures, relative to Tat alone. Further, genes associated with cytoskeleton dynamics in inflammatory microglia were downregulated by 4OI treatment. Together, the results strongly suggest 4-octyl-itaconate holds promise as a potential candidate for therapeutic development aimed at addressing HAND coupled with CUD comorbidities.

Heparan sulfate modifications of betaglycan promote TIMP3-dependent ectodomain shedding to fine-tune TGF-ß signaling.

In pathologies such as cancer, aberrant Transforming Growth Factor-ß (TGF-ß) signaling exerts profound tumor intrinsic and extrinsic consequences. Intense clinical endeavors are underway to target this pivotal pathway. Central to the success of these interventions is pinpointing factors that decisively modulate the TGF-ß responses. Betaglycan/type III TGF-ß receptor (TßRIII), is an established co-receptor for the TGF-ß superfamily known to bind directly to TGF-ßs 1-3 and inhibin A/B. While betaglycan can be membrane-bound, it can also undergo ectodomain cleavage to produce soluble-betaglycan that can sequester its ligands. The extracellular domain of betaglycan undergoes heparan sulfate and chondroitin sulfate glycosaminoglycan modifications, transforming betaglycan into a proteoglycan. Here we report the unexpected discovery that the heparan sulfate modifications are critical for the ectodomain shedding of betaglycan. In the absence of such modifications, betaglycan is not shed. Such shedding is indispensable for the ability of betaglycan to suppress TGF-ß signaling and the cells' responses to exogenous TGF-ß ligands. Using unbiased transcriptomics, we identified TIMP3 as a key regulator of betaglycan shedding and thereby TGF-ß signaling. Our results bear significant clinical relevance as modified betaglycan is present in the ascites of patients with ovarian cancer and can serve as a marker for predicting patient outcomes and TGF-ß signaling responses. These studies are the first to demonstrate a unique reliance on the glycosaminoglycan modifications of betaglycan for shedding and influence on TGF-ß signaling responses. Dysregulated shedding of TGF-ß receptors plays a vital role in determining the response and availability of TGF-ßs', which is crucial for prognostic predictions and understanding of TGF-ß signaling dynamics.

Structure-activity and mechanistic studies of non-peptidic inhibitors of the PLK1 polo box domain identified through REPLACE.

Polo-like kinase 1 (PLK1) is a serine/threonine-protein kinase involved in cell cycle regulation and mitotic progression. Studies have shown that PLK1 is upregulated in many tumors and high levels are adversely related to a poor prognosis. Knocking down or inhibiting PLK1 results in synthetic lethality in PTEN deficient prostate tumors and Kras mutant colorectal tumors, further validating PLK1 as an oncotarget. Substrate recognition by PLK1 occurs through the Polo-Box Domain (PBD), which is a phospho-peptide binding site also responsible for subcellular localization. Much effort has been directed to target this kinase therapeutically through the ATP-binding site, and a few such inhibitors have advanced to clinical trials however with limited clinical efficacy. Moreover, it has been shown that a point mutation in PLK1 (C67V) confers dramatic cellular resistance to catalytic site inhibitors. An alternative approach to target PLK1 potently and selectively is through the PBD to block its protein-protein interactions. Through the REPLACE strategy, for converting peptide inhibitors into more drug-like non peptidic compounds, a PBD targeting compound series ("ABBAs"), has been identified and the key determinants of potency and selectivity elucidated through structure-activity relationship studies. In cellular experiments, the ABBAs were shown to lead to profound effects on the cell cycle, to inhibit tumor proliferation and overcome resistance of cells expressing the PLK1 C67V mutant to ATP-based inhibitors. These non-ATP competitive inhibitors of PLK1 were also used chemical biology probes to investigate the gene regulatory effects of PLK1, known to act on transcription factors such as p53.

Reciprocal SOX2 regulation by SMAD1-SMAD3 is critical for anoikis resistance and metastasis in cancer.

Growth factors in tumor environments are regulators of cell survival and metastasis. Here, we reveal the dichotomy between TGF-ß superfamily growth factors BMP and TGF-ß/activin and their downstream SMAD effectors. Gene expression profiling uncovers SOX2 as a key contextual signaling node regulated in an opposing manner by BMP2, -4, and -9 and TGF-ß and activin A to impact anchorage-independent cell survival. We find that SOX2 is repressed by BMPs, leading to a reduction in intraperitoneal tumor burden and improved survival of tumor-bearing mice. Repression of SOX2 is driven by SMAD1-dependent histone H3K27me3 recruitment and DNA methylation at SOX2's promoter. Conversely, TGF-ß, which is elevated in patient ascites, and activin A can promote SOX2 expression and anchorage-independent survival by SMAD3-dependent histone H3K4me3 recruitment. Our findings identify SOX2 as a contextual and contrastingly regulated node downstream of TGF-ß members controlling anchorage-independent survival and metastasis in ovarian cancers.

HPV-inactive cell populations arise from HPV16-transformed human keratinocytes after p53 knockout.

HPV-inactive head and neck and cervical cancers contain HPV DNA but do not express HPV E6/E7. HPV-positive primary head and neck tumors usually express E6/E7, however they may produce HPV-inactive metastases. These observations led to our hypothesis that HPV-inactive cancers begin as HPV-active lesions, losing dependence on E6/E7 expression during progression. Because HPV-inactive cervical cancers often have mutated p53, we investigated whether p53 loss may play a role in the genesis of HPV-inactive cancers. p53 knockout (p53-KO) by CRISPR-Cas9 resulted in a 5-fold reduction of E7 mRNA in differentiation-resistant HPV16 immortalized human keratinocytes (HKc/DR). E7 expression was restored by 5-Aza-2 deoxycytidine in p53 KO lines, suggesting a role of DNA methylation in this process. In-situ hybridization showed that p53 KO lines consist of mixed populations of E6/E7-positive and negative cells. Hence, loss of p53 predisposes HPV16 transformed cells to losing dependence on the continuous expression of HPV oncogenes for proliferation.

Self-assembling 3D spheroid cultures of human neonatal keratinocytes have enhanced regenerative properties.

Relative to conventional two-dimensional (2-D) culture, three-dimensional (3-D) suspension culture of epithelial cells more closely mimics the in vivo cell microenvironment regarding cell architecture, cell to matrix interaction, and osmosis exchange. However, primary normal human keratinocytes (NHKc) rapidly undergo terminal differentiation and detachment-induced cell death (anoikis) upon disconnection from the basement membrane, thus greatly constraining their use in 3-D suspension culture models. Here, we examined the 3-D anchorage-free growth potential of NHKc isolated from neonatal skin explants of 59 different individuals. We found that 40% of all isolates naturally self-assembled into multicellular spheroids within 24 h in anchorage-free culture, while 60% did not. Placing a single spheroid back into 2-D monolayer culture yielded proliferating cells that expressed elevated levels of nuclear P63 and basal cytokeratin 14. These cells also displayed prolonged keratinocyte renewal and a gene expression profile corresponding to cellular heterogeneity, quiescence, and de-differentiation. Notably, spheroid-derived (SD) NHKc were enriched for a P63/K14 double-positive population that formed holoclonal colonies and reassembled into multicellular spheroids during 3-D suspension subculture. This study reveals marked phenotypic differences in neonatal keratinocyte suspension cultures isolated from different individuals andpresenta model system that can be readily employed to study epithelial cell behavior, along with a variety of dermatological diseases.