Evidence supporting the oncogenic role of BAZ1B in colorectal cancer.

Bromodomain Adjacent to Zinc Finger Domain 1B (BAZ1B) is involved in multiple nuclear processes, and its role in tumorigenesis is emerging. However, the function of BAZ1B in colorectal cancer (CRC) remains largely unexplored. High-density tissue microarrays comprising 100 pairs of matched normal colon and treatment-naïve CRC samples were analyzed by immunohistochemistry with an anti-BAZ1B antibody. The HCT116 and SW480 CRC cell lines were used for overexpression and small hairpin RNA-mediated BAZ1B knockdown models, respectively. Both cell lines were xenografted to immunodeficient NU/J mice to assess tumor burden. The molecular consequences of alterations of BAZ1B expression were assessed by RNA-Seq of xenografts and functional analyses using the Reactome database. Immunohistochemical analysis of BAZ1B showed that BAZ1B staining intensity was higher in 93 tumor specimens and significantly correlated with tumor size (P = 0.03), but not with the presence of KRAS mutation. BAZ1B overexpression significantly increased and its knockdown inhibited the proliferation of HCT116 and SW480 cell lines, respectively. These findings were reproduced when both cell lines were grown as xenografts. RNA-Seq of HCT116 and SW480 xenografts identified 2046 and 99 differentially expressed genes (DEGs) (adjusted P ≤ 0.05), respectively. Functional annotation of DEGs identified already established as well as new molecular processes dependent on BAZ1B protein expression. In conclusion, BAZ1B is overexpressed in CRC tissue and contributes to CRC cell proliferation in vitro and in vivo. The data support the emerging oncogenic role of BAZ1B in cancerogenesis including in CRC.

Oncology Drug Repurposing for Sepsis Treatment.

Sepsis involves life-threatening organ dysfunction caused by a dysregulated host response to infection. Despite three decades of efforts and multiple clinical trials, no treatment, except antibiotics and supportive care, has been approved for this devastating syndrome. Simultaneously, numerous preclinical studies have shown the effectiveness of oncology-indicated drugs in ameliorating sepsis. Here we focus on cataloging these efforts with both oncology-approved and under-development drugs that have been repositioned to treat bacterial-induced sepsis models. In this context, we also envision the exciting prospect for further standard and oncology drug combination testing that could ultimately improve clinical outcomes in sepsis.

Selective Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) Inhibition by the SCH772984 Compound Attenuates In Vitro and In Vivo Inflammatory Responses and Prolongs Survival in Murine Sepsis Models.

Sepsis is the leading cause of death in intensive care units worldwide. Current treatments of sepsis are largely supportive and clinical trials using specific pharmacotherapy for sepsis have failed to improve outcomes. Here, we used the lipopolysaccharide (LPS)-stimulated mouse RAW264.7 cell line and AlphaLisa assay for TNFa as a readout to perform a supervised drug repurposing screen for sepsis treatment with compounds targeting epigenetic enzymes, including kinases. We identified the SCH772984 compound, an extracellular signal-regulated kinase (ERK) 1/2 inhibitor, as an effective blocker of TNFa production in vitro. RNA-Seq of the SCH772984-treated RAW264.7 cells at 1, 4, and 24 h time points of LPS challenge followed by functional annotation of differentially expressed genes highlighted the suppression of cellular pathways related to the immune system. SCH772984 treatment improved survival in the LPS-induced lethal endotoxemia and cecal ligation and puncture (CLP) mouse models of sepsis, and reduced plasma levels of Ccl2/Mcp1. Functional analyses of RNA-seq datasets for kidney, lung, liver, and heart tissues from SCH772984-treated animals collected at 6 h and 12 h post-CLP revealed a significant downregulation of pathways related to the immune response and platelets activation but upregulation of the extracellular matrix organization and retinoic acid signaling pathways. Thus, this study defined transcriptome signatures of SCH772984 action in vitro and in vivo, an agent that has the potential to improve sepsis outcome.

Inhibition of PIM Kinases in DLBCL Targets MYC Transcriptional Program and Augments the Efficacy of Anti-CD20 Antibodies.

The family of PIM serine/threonine kinases includes three highly conserved oncogenes, PIM1, PIM2, and PIM3, which regulate multiple prosurvival pathways and cooperate with other oncogenes such as MYC. Recent genomic CRISPR-Cas9 screens further highlighted oncogenic functions of PIMs in diffuse large B-cell lymphoma (DLBCL) cells, justifying the development of small-molecule PIM inhibitors and therapeutic targeting of PIM kinases in lymphomas. However, detailed consequences of PIM inhibition in DLBCL remain undefined. Using chemical and genetic PIM blockade, we comprehensively characterized PIM kinase-associated prosurvival functions in DLBCL and the mechanisms of PIM inhibition-induced toxicity. Treatment of DLBCL cells with SEL24/MEN1703, a pan-PIM inhibitor in clinical development, decreased BAD phosphorylation and cap-dependent protein translation, reduced MCL1 expression, and induced apoptosis. PIM kinases were tightly coexpressed with MYC in diagnostic DLBCL biopsies, and PIM inhibition in cell lines and patient-derived primary lymphoma cells decreased MYC levels as well as expression of multiple MYC-dependent genes, including PLK1. Chemical and genetic PIM inhibition upregulated surface CD20 levels in an MYC-dependent fashion. Consistently, MEN1703 and other clinically available pan-PIM inhibitors synergized with the anti-CD20 monoclonal antibody rituximab in vitro, increasing complement-dependent cytotoxicity and antibody-mediated phagocytosis. Combined treatment with PIM inhibitor and rituximab suppressed tumor growth in lymphoma xenografts more efficiently than either drug alone. Taken together, these results show that targeting PIM in DLBCL exhibits pleiotropic effects that combine direct cytotoxicity with potentiated susceptibility to anti-CD20 antibodies, justifying further clinical development of such combinatorial strategies. SIGNIFICANCE: These findings demonstrate that inhibition of PIM induces DLBCL cell death via MYC-dependent and -independent mechanisms and enhances the therapeutic response to anti-CD20 antibodies by increasing CD20 expression.

Cytotoxic Efficacy and Resistance Mechanism of a TRAIL and VEGFA-Peptide Fusion Protein in Colorectal Cancer Models.

TNF-related apoptosis-inducing ligand (TRAIL) is a type II transmembrane protein capable of selectively inducing apoptosis in cancer cells by binding to its cognate receptors. Here, we examined the anticancer efficacy of a recently developed chimeric AD-O51.4 protein, a TRAIL fused to the VEGFA-originating peptide. We tested AD-O51.4 protein activity against human colorectal cancer (CRC) models and investigated the resistance mechanism in the non-responsive CRC models. The quantitative comparison of apoptotic activity between AD-O51.4 and the native TRAIL in nine human colorectal cancer cell lines revealed dose-dependent toxicity in seven of them; the immunofluorescence-captured receptor abundance correlated with the extent of apoptosis. AD-O51.4 reduced the growth of CRC patient-derived xenografts (PDXs) with good efficacy. Cell lines that acquired AD-O51.4 resistance showed a significant decrease in surface TRAIL receptor expression and apoptosis-related proteins, including Caspase-8, HSP60, and p53. These results demonstrate the effectiveness of AD-O51.4 protein in CRC preclinical models and identify the potential mechanism underlying acquired resistance. Progression of AD-O51.4 to clinical trials is expected.

Functional analyses of a low-penetrance risk variant rs6702619/1p21.2 associating with colorectal cancer in Polish population.

Several studies employed the genome-wide association (GWA) analysis of single-nucleotide polymorphisms (SNPs) to identify susceptibility regions in colorectal cancer (CRC). However, the functional studies exploring the role of associating SNPs with cancer biology are limited. Herein, using chromatin immunoprecipitation assay (ChIP), reporter assay and chromosome conformation capture sequencing (3C-Seq) augmented with publically available genomic and epigenomic databases we aimed to define the function of rs6702619/1p21.2 region associated with CRC in the Polish population. Using ChIP we confirmed that rs6702619 region is occupied by a CTCF, a master regulator of long-range genomic interactions, and is decorated with enhancer-like histone modifications. The enhancer blocking assay revealed that rs6702619 region acts as an insulator with activity dependent on the SNP genotype. Finally, a 3C-Seq survey indicated more than a hundred loci in the rs6702619 locus interactome, including GNAS gene that is frequently amplified in CRC. Taken together, we showed that the CRC-associated rs6702619 region has in vitro and in vivo properties of an insulator that demonstrates long-range physical interactions with CRC-relevant loci.

Challenges in Stratifying the Molecular Variability of Patient-Derived Colon Tumor Xenografts.

Colorectal cancer (CRC) is the second most common cancer in Europe and a leading cause of death worldwide. Patient-derived xenograft (PDX) models maintain complex intratumoral biology and heterogeneity and therefore remain the platform of choice for translational drug discovery. In this study, we implanted 37 primary CRC tumors and five CRC cell lines into NU/J mice to develop xenograft models. Primary tumors and established xenografts were histologically assessed and surveyed for genetic variants and gene expression using a panel of 409 cancer-related genes and RNA-seq, respectively. More than half of CRC tumors (20 out of 37, 54%) developed into a PDX. Histological assessment confirmed that PDX grading, stromal components, inflammation, and budding were consistent with those of the primary tumors. DNA sequencing identified an average of 0.14 variants per gene per sample. The percentage of mutated variants in PDXs increased with successive passages, indicating a decrease in clonal heterogeneity. Gene Ontology analyses of 4180 differentially expressed transcripts (adj. p value < 0.05) revealed overrepresentation of genes involved in cell division and catabolic processes among the transcripts upregulated in PDXs; downregulated transcripts were associated with GO terms related to extracellular matrix organization, immune responses, and angiogenesis. Neither a transcriptome-based consensus molecular subtype (CMS) classifier nor three other predictors reliably matched PDX molecular subtypes with those of the primary tumors. In sum, both genetic and transcriptomic profiles differed between donor tumors and PDXs, likely as a consequence of subclonal evolution at the early phase of xenograft development, making molecular stratification of PDXs challenging.

Downregulation of PTPRH (Sap-1) in colorectal tumors.

Tyrosine phosphorylation is one of the basic mechanisms for signal transduction in the cell. Receptors exhibiting tyrosine kinase activity are widely involved in carcinogenesis and are negatively regulated by receptor protein tyrosine phosphatases (RPTP). Genes encoding different RPTPs are affected by aberrant epigenetic regulation in cancer. PTPRH (SAP-1) has been previously described to be overexpressed in colorectal cancer (CRC) and classified as an oncogenic factor. Previous microarray-based mRNA expression comparison of colorectal adenomas (AD), CRC and normal mucosa samples (NM) demonstrated that PTPRH tumor expression is the most reduced of all RPTP genes. qRT-PCR validation revealed gene downregulation for CRC (7.6-fold-change; P<0.0001) and AD (3.4-fold-change; P<0.0001) compared to NM. This was confirmed by immunohistochemical staining of tumor and NM sections as pronounced decrease of protein expression was observed in CRCs compared to the corresponding normal tissue. DNA methylation of two PTPRH promoter fragments was analyzed by pyrosequencing in a group of CRC, and AD patients as well as NM samples and CRC cell lines. The mean DNA methylation levels of these two regions were significantly higher in CRC than in NM. Both regions were highly methylated in SW480 and HCT116 cell lines contrary to unmethylated HT29 and COLO205. Cell lines with highly methylated promoters notably showed lower PTPRH expression levels, lower RNA II polymerase concentrations and higher levels of H3K27 trimethylation in the promoter and gene body, measured by chromatin immunoprecipitation. Cells were cultured with 5-aza-deoxycitidine and an increase in PTPRH expression was observed in SW480 and HCT116, whereas this was unchanged in the unmethylated cell lines. The results indicate that PTPRH is downregulated in colorectal tumors and its expression is epigenetically regulated via DNA methylation and chromatin modifications.

SEL120-34A is a novel CDK8 inhibitor active in AML cells with high levels of serine phosphorylation of STAT1 and STAT5 transactivation domains.

Inhibition of oncogenic transcriptional programs is a promising therapeutic strategy. A substituted tricyclic benzimidazole, SEL120-34A, is a novel inhibitor of Cyclin-dependent kinase 8 (CDK8), which regulates transcription by associating with the Mediator complex. X-ray crystallography has shown SEL120-34A to be a type I inhibitor forming halogen bonds with the protein's hinge region and hydrophobic complementarities within its front pocket. SEL120-34A inhibits phosphorylation of STAT1 S727 and STAT5 S726 in cancer cells in vitro. Consistently, regulation of STATs- and NUP98-HOXA9- dependent transcription has been observed as a dominant mechanism of action in vivo. Treatment with the compound resulted in a differential efficacy on AML cells with elevated STAT5 S726 levels and stem cell characteristics. In contrast, resistant cells were negative for activated STAT5 and revealed lineage commitment. In vivo efficacy in xenotransplanted AML models correlated with significant repression of STAT5 S726. Favorable pharmacokinetics, confirmed safety and in vivo efficacy provide a rationale for the further clinical development of SEL120-34A as a personalized therapeutic approach in AML.

Prenyl Ammonium Salts--New Carriers for Gene Delivery: A B16-F10 Mouse Melanoma Model.

PURPOSE: Prenyl ammonium iodides (Amino-Prenols, APs), semi-synthetic polyprenol derivatives were studied as prospective novel gene transfer agents. METHODS: AP-7, -8, -11 and -15 (aminoprenols composed of 7, 8, 11 or 15 isoprene units, respectively) were examined for their capacity to form complexes with pDNA, for cytotoxicity and ability to transfect genes to cells. RESULTS: All the carriers were able to complex DNA. The highest, comparable to commercial reagents, transfection efficiency was observed for AP-15. Simultaneously, AP-15 exhibited the lowest negative impact on cell viability and proliferation--considerably lower than that of commercial agents. AP-15/DOPE complexes were also efficient to introduce pDNA to cells, without much effect on cell viability. Transfection with AP-15/DOPE complexes influenced the expression of a very few among 44 tested genes involved in cellular lipid metabolism. Furthermore, complexes containing AP-15 and therapeutic plasmid, encoding the TIMP metallopeptidase inhibitor 2 (TIMP2), introduced the TIMP2 gene with high efficiency to B16-F10 melanoma cells but not to B16-F10 melanoma tumors in C57BL/6 mice, as confirmed by TIMP2 protein level determination. CONCLUSION: Obtained results indicate that APs have a potential as non-viral vectors for cell transfection.

Beads-free protein immunoprecipitation for a mass spectrometry-based interactome and posttranslational modifications analysis.

BACKGROUND: Protein immunoprecipitation (IP) coupled with MS provides means to interrogate protein complexes and their posttranslational modifications (PTMs). In a typical protein IP assay antibodies are conjugated to protein A/G beads requiring large amounts of antibodies, tube transfers and centrifugations. RESULTS: As an alternative, we present Matrix-IP, beads-free microplate-based platform with surface-immobilized antibodies. Assay utilizes standard 96-well polypropylene PCR plates that are laboratory-fabricated with UV-C light and then protein A/G coated prior to IP reaction. We demonstrate application of Matrix-IP platform in MS analysis of heterogeneous nuclear ribonucleoprotein K (hnRNP K) interactome and PTMs. CONCLUSION: Matrix-IP is time-saving, easy to use high throughput method adaptable for low sample amounts and automation.

Epigenetic-Mediated Downregulation of µ-Protocadherin in Colorectal Tumours.

Carcinogenesis involves altered cellular interaction and tissue morphology that partly arise from aberrant expression of cadherins. Mucin-like protocadherin is implicated in intercellular adhesion and its expression was found decreased in colorectal cancer (CRC). This study has compared MUPCDH (CDHR5) expression in three key types of colorectal tissue samples, for normal mucosa, adenoma, and carcinoma. A gradual decrease of mRNA levels and protein expression was observed in progressive stages of colorectal carcinogenesis which are consistent with reports of increasing MUPCDH 5' promoter region DNA methylation. High MUPCDH methylation was also observed in HCT116 and SW480 CRC cell lines that revealed low gene expression levels compared to COLO205 and HT29 cell lines which lack DNA methylation at the MUPCDH locus. Furthermore, HCT116 and SW480 showed lower levels of RNA polymerase II and histone H3 lysine 4 trimethylation (H3K4me3) as well as higher levels of H3K27 trimethylation at the MUPCDH promoter. MUPCDH expression was however restored in HCT116 and SW480 cells in the presence of 5-Aza-2'-deoxycytidine (DNA methyltransferase inhibitor). Results indicate that µ-protocadherin downregulation occurs during early stages of tumourigenesis and progression into the adenoma-carcinoma sequence. Epigenetic mechanisms are involved in this silencing.

Regulation of the expression of claudin 23 by the enhancer of zeste 2 polycomb group protein in colorectal cancer.

Altered epigenetic mechanisms, similar to gene mutations, contribute to the pathogenesis and molecular heterogeneity of neoplasms, including colorectal cancer (CRC). Enhancer of zeste 2 (EZH2) is a histone methyltransferase, which is involved in epigenetic gene silencing and is aberrantly expressed in CRC. Therefore, the identification of the genes regulated by EZH2 in CRC is important to improve current understanding of its role in cancer epigenetics. The present study used chromatin immunoprecipitation (ChIP) followed by deep sequencing to assess genome-wide EZH2­DNA interactions in healthy or CRC mucosa samples. In total, 86.9/61.6 and 92.5/62.6 million tags were sequenced/mapped in healthy and CRC mucosa samples, respectively. The EZH2-binding densities were correlated with transcriptomic datasets and this demonstrated that the claudin-23 (CLDN23) gene, which encodes a component of cell-cell adhesion structures, was occupied by EZH2 and significantly silenced in CRC tissue. The measurement of DNA methylation at the CLDN23 promoter using pyrosequencing excluded the possibility that silencing of this gene in CRC patient samples was a result of DNA hypermethylation. Following treatment of the Colo205 and HT-29 CRC cell lines, with the EZH2 inhibitor, GSK126, the level of histone H3 lysine 27 trimethylation (H3K27me3) was reduced and the mRNA and protein expression levels of CLDN23 were increased. ChIP analysis confirmed that the level of H3K27m3 along the CLDN23 gene was decreased in the GSK126-treated cell lines. Furthermore, ChIP analysis of these samples detected histone H3 lysine 4 trimethylation (H3K4me3) at the CLDN23 promoter, demonstrating that the balance between H3K27me3 and H3K4me3 may underlie the regulation of the expression of CLDN23. The present study demonstrated an epigenetic link between the activity of the EZH2 methyltransferase at the CLDN23 locus and the expression of CLDN23 in CRC tissue.

The role of the SHH gene in prostate cancer cell resistance to paclitaxel.

BACKGROUND: The increased activity of the Sonic Hedgehog (SHH) pathway has been demonstrated in many types of cancer including prostate cancer (PCa). It has been shown that SHH pathway is involved in tumor angiogenesis and in regulation of metabolism of cancer stem cells. The increased activity of the SHH pathway is responsible for generation and maintenance of the multidrug resistance in cancer cells. A key role in the development of this insensitivity to cytotoxic drugs play ATP-binding cassette (ABC) transporters. METHODS: SHH encoding plasmid was stably transfected into PCa cell lines DU145 and LNCaP. The expression of SHH was silenced by shRNA and the level of SHH was tested by quantitative (q)PCR and western blot methods. The effect of SHH overexpression in cells after treatment with paclitaxel was measured by MTT assay, crystal violet assay and flow cytometry. The level of 44 ABC transporters was estimated by qPCR. RESULTS: Expression of exogenous SHH protein in DU145 and LNCaP cell lines enhanced their resistance to paclitaxel along with increased expression of ABC transporters transcripts. Paclitaxel treatment further enhanced the expression of increased ABC transporters transcripts in cells overexpressing SHH. CONCLUSIONS: Overexpression of SHH enhances PCa cell lines resistance to paclitaxel. Higher level of SHH leads to increase in ABC transporters expression in a manner dependent on paclitaxel.

Maize calcium-dependent protein kinase (ZmCPK11): local and systemic response to wounding, regulation by touch and components of jasmonate signaling.

Expression of ZmCPK11, a member of the maize (Zea mays) calcium-dependent protein kinases (CDPKs) family, is induced by mechanical wounding. A rapid increase of the activity of a 56-kDa CDPK has been observed in damaged leaves. In the present work, it is shown that the 56-kDa CDPK, identified as ZmCPK11, is also activated in non-wounded leaves as an element of systemic wound response. Moreover, an increase of the enzyme's activity and induction of ZmCPK11 expression was observed after touching the leaves. To study the role of ZmCPK11 in wound and touch signaling, transgenic Arabidopsis thaliana plants in which c-Myc-ZmCPK11 was expressed under control of the CaMV 35S promoter were generated. Analysis of the transgenic plants showed that c-Myc-ZmCPK11 was activated upon wounding and touching. Furthermore, pre-treatment with acetylsalicylic acid (acSA), an inhibitor of jasmonic acid (JA)-dependent wound signaling, abolished the wound-induced activation of ZmCPK11 in maize and the transgenic A. thaliana plants. Methyl jasmonate (MeJA) and linolenic acid (LA) stimulated the activity of ZmCPK11 as well as induced the expression of ZmCPK11 and other wound-responsive genes, lipoxygenase 1 (ZmLOX1) and proteinase inhibitor 1 (ZmWIP1). These results indicate that ZmCPK11, regulated at the enzymatic and transcriptional level by LA and MeJA, is a component of touch- and wound-induced pathway(s), participating in early stages of local and systemic responses.