Mutation and expression analysis of the IDH1, IDH2, DNMT3A, and MYD88 genes in colorectal cancer

Colorectal cancer (CRC) is one of the leading causes of death around the world. Its genetic mechanism was intensively investigated in the past decades with findings of a number of canonical oncogenes and tumor-suppressor genes such as APC, KRAS, and TP53. Recent genome-wide association and sequencing studies have identified a series of promising oncogenes including IDH1, IDH2, DNMT3A, and MYD88 in hematologic malignancies. However, whether these genes are involved in CRC remains unknown. In this study, we screened the hotspot mutations of these four genes in 305 CRC samples from Han Chinese by direct sequencing. mRNA expression levels of these genes were quantified by quantitative real-time PCR (RT-qPCR) in paired cancerous and paracancerous tissues. Association analyses between mRNA expression levels and different cancerous stages were performed. Except for one patient harboring IDH1 mutation p.I99M, we identified no previously reported hotspot mutations in colorectal cancer tissues. mRNA expression levels of IDH1, DNMT3A, and MYD88, but not IDH2, were significantly decreased in the cancerous tissues comparing with the paired paracancerous normal tissues. Taken together, the hotspot mutations of IDH1, IDH2, DNMT3A, and MYD88 gene were absent in CRC. Aberrant mRNA expression of IDH1, DNMT3A, and MYD88 gene might be actively involved in the development of CRC.     

DNA Methylation and Carcinogenesis

The hypothesis of the exclusively genetic origin of cancer (cancer is a disease of genes, a tumor without any damage to the genome does not exist) dominated in the oncology until recently. A considerable amount of data confirming this hypothesis was accumulated during the last quarter of the last century. It was demonstrated that the accumulation of damage of specific genes lies at the origin of a tumor and its following progression. The damage gives rise to structural changes in the respective proteins and, consequently, to inappropriate mitogenic stimulation of cells (activation of oncogenes) or to the inactivation of tumor suppressor genes that inhibit cell division, or to the combination of both (in most cases). According to an alternative (epigenetic) hypothesis that was extremely unpopular until recently, a tumor is caused not by a gene damage, but by an inappropriate function of genes (cancer is a disease of gene regulation and differentiation). However, recent studies led to the convergence of these hypotheses that initially seemed to be contradictory. It was established that both factors–genetic and epigenetic–lie at the origin of carcinogenesis. The relative contribution of each varies significantly in different human tumors. Suppressor genes and genes of repair are inactivated in tumors due to their damage or methylation of their promoters (in the latter case an epimutation, an epigenetic equivalent of a mutation, occurs, producing the same functional consequences). It is becoming evident that not only the mutagens, but various factors influencing cell metabolism, notably methylation, should be considered as carcinogens.     

Mutation analysis of large tumor suppressor genes LATS1 and LATS2 supports a tumor suppressor role in human cancer

In recent years, human cancer genome projects provide unprecedented opportunities for the discovery of cancer genes and signaling pathways that contribute to tumor development. While numerous gene mutations can be identified from each cancer genome, what these mutations mean for cancer is a challenging question to address, especially for those from less understood putative new cancer genes. As a powerful approach, in silico bioinformatics analysis could efficiently sort out mutations that are predicted to damage gene function. Such an analysis of human large tumor suppressor genes, LATS1 and LATS2, has been carried out and the results support a role of hLATS1//2 as negative growth regulators and tumor suppressors.     

Addiction to protein kinase CK2: A common denominator of diverse cancer cells?

At variance with most oncogenic protein kinases whose malignancy is generally due to genetic alterations conferring constitutive activity, CK2 is a highly pleiotropic Ser/Thr protein kinase naturally endowed with constitutive activity and lacking gain-of-function mutants. Nonetheless CK2 is abnormally elevated in a wide variety of tumors and there is strong evidence that it operates as a cancer driver by creating a cellular environment favorable to neoplasia: notably, CK2 plays a global role as an anti-apoptotic and pro-survival agent, it enhances the multi-drug resistance (MDR) phenotype, it assists the chaperone machinery which protects the “onco-kinome” and it promotes neo-angiogenesis. Based on this scenario we propose that the implication of CK2 in neoplasia is an example of “non oncogene addiction”, i.e. over reliance of the perturbed cellular signaling network on high CK2 level for its own maintenance. Consistent with this, an ample spectrum of diverse types of cancer cells have been already shown to rely on high CK2 level for their survival, as judged from their response to specific CK2 inhibitors and silencing of endogenous CK2 catalytic subunits. Remarkably, among these are cells whose cancer phenotype arises from the genetic alteration of onco-kinases (e.g. Abl and Alk) different from CK2 and insensitive to the CK2 inhibitors used in those experiments. Based on these premises, CK2 could represent a “multi-purpose” target for the treatment of different kinds of tumors.     

The RNA expression signature of the HepG2 cell line as determined by the integrated analysis of miRNA and mRNA expression profiles

Understanding miRNAs' regulatory networks and target genes could facilitate the development of therapies for human diseases such as cancer. Although much useful gene expression profiling data for tumor cell lines is available, microarray data for miRNAs and mRNAs in the human HepG2 cell line have only been compared with that of other cell lines separately. The relationship between miRNAs and mRNAs in integrated expression profiles for HepG2 cells is still unknown. To explore the miRNA–mRNA correlations in hepatocellular carcinoma (HCC) cells, we performed miRNA and mRNA expression profiling in HepG2 cells and normal liver HL-7702 cells at the genome scale using next-generation sequencing technology. We identified 193 miRNAs that are differentially expressed in these two cell lines. Of these, 89 miRNAs were down-regulated in HepG2 cells compared with HL-7702 cells, while 104 miRNAs were up-regulated. We also observed 3035 mRNAs that are significantly dys-regulated in HepG2 cells. We then performed an integrated analysis of the expression data for differentially expressed miRNAs and mRNAs and found several miRNA–mRNA pairs that are significantly correlated in HepG2 cells. Further analysis suggested that these differentially expressed genes were enriched in four tumorigenesis-related signaling pathways, namely, ErbB, JAK–STAT, mTOR, and WNT, which until now had not been fully reported. Our results could be helpful in understanding the mechanisms of HCC occurrence and development.     

Microarray profiling of HepG2 cells ectopically expressing NDRG2

Previous studies have demonstrated that N-Myc downstream-regulated gene 2 (NDRG2) is a tumor suppressor that is downregulated in many human cancers and when overexpressed, can inhibit tumor growth and metastasis. However, its molecular function, its modulatory targets, and signaling pathways associated with it remain unclear. Here, in an effort to identify the genes modulated by NDRG2 expression, a microarray study was conducted to detect the expression profile of HepG2 cells overexpressing NDRG2 or LacZ. Gene Ontology (GO) biological process analysis revealed that genes related to G protein signaling pathway were upregulated. Five of them were selected and verified by real-time PCR. Gene sets related to M phase of cell cycle were downregulated. This was in agreement with cell cycle analysis. Signaling pathway analysis demonstrated apparent augmented hematopoietic cell lineage pathway and cell adhesion, but reduced glycosylphosphatidylinositol (GPI)-anchor biosynthesis, protein degradation and SNARE interactions. Furthermore, through motif analysis and experimental validation, we found that the p38 phosphorylation can be increased by NDRG2. Our research provides the molecular basis for understanding the role of NDRG2 in tumor cells and raises interesting questions about its mechanisms and potential use in cancer therapy.     

The two faces of PTP1B in cancer

PTP1B is a classical non-transmembrane protein tyrosine phosphatase that plays a key role in metabolic signaling and is a promising drug target for type 2 diabetes and obesity. Accumulating evidence also indicates that PTP1B is involved in cancer, but contrasting findings suggest that it can exert both tumor suppressing and tumor promoting effects depending on the substrate involved and the cellular context. In this review, we will discuss the diverse mechanisms by which PTP1B may influence tumorigenesis as well as recent in vivo data on the impact of PTP1B deficiency in murine cancer models. Together, these results highlight not only the great potential of PTP1B inhibitors in cancer therapy but also the need for a better understanding of PTP1B function prior to use of these compounds in human patients.     

δ-Opioid receptor-stimulated Akt signaling in neuroblastoma × glioma (NG108-15) hybrid cells involves receptor tyrosine kinase-mediated PI3K activation

δ-Opioid receptor (DOR) agonists possess cytoprotective properties, an effect associated with activation of the “pro-survival” kinase Akt. Here we delineate the signal transduction pathway by which opioids induce Akt activation in neuroblastoma × glioma (NG108-15) hybrid cells. Exposure of the cells to both [D-Pen^2,5]enkephalin and etorphine resulted in a time- and dose-dependent increase in Akt activity, as measured by means of an activation-specific antibody recognizing phosphoserine-473. DOR-mediated Akt signaling is blocked by the opioid antagonist naloxone and involves inhibitory G_i/o proteins, because pre-treatment with pertussis toxin, but not over-expression of the G_q/11 scavengers EBP50 and GRK2-K220R, prevented this effect. Further studies with Wortmannin and LY294002 revealed that phophoinositol-3-kinase (PI3K) plays a central role in opioid-induced Akt activation. Opioids stimulate Akt activity through transactivation of receptor tyrosine kinases (RTK), because pre-treatment of the cells with inhibitors for neurotrophin receptor tyrosine kinases (AG879) and the insulin-like growth factor receptor IGF-1 (AG1024), but not over-expression of the Gβγ scavenger phosducin, abolished this effect. Activated Akt translocates to the nuclear membrane, where it promotes GSK3 phosphorylation and prevents caspase-3 cleavage, two key events mediating inhibition of cell apoptosis and enhancement of cell survival. Taken together, these results demonstrate that in NG108-15 hybrid cells DOR agonists possess cytoprotective properties mediated by activation of the RTK/PI3K/Akt signaling pathway.     

Systematic pharmacological analysis of agonistic and antagonistic fibroblast growth factor receptor 1 MAbs reveals a similar unique mode of action

Fibroblast growth factor receptor 1 (FGFR1) is a receptor tyrosine kinase that plays a major role in developmental processes and metabolism. The dysregulation of FGFR1 through genetic aberrations leads to skeletal and metabolic diseases as well as cancer. For this reason, FGFR1 is a promising therapeutic target, yet a very challenging one due to potential on-target toxicity. More puzzling is that both agonistic and antagonistic FGFR1 antibodies are reported to exhibit similar toxicity profiles in vivo, namely weight loss. In this study, we aimed to assess and compare the mechanism of action of these molecules to better understand this apparent contradiction. By systematically comparing the binding of these antibodies and the activation or the inhibition of the major FGFR1 signaling events, we demonstrated that the molecules displayed similar properties and can behave either as an agonist or antagonist depending on the presence or the absence of the endogenous ligand. We further demonstrated that these findings translated in xenografts mice models. In addition, using time-resolved FRET and mass spectrometry analysis, we showed a functionally distinct FGFR1 active conformation in the presence of an antibody that preferentially activates the FGFR substrate 2 (FRS2)-dependent signaling pathway, demonstrating that modulating the geometry of a FGFR1 dimer can effectively change the signaling outputs and ultimately the activity of the molecule in preclinical studies. Altogether, our results highlighted how bivalent antibodies can exhibit both agonistic and antagonistic activities and have implications for targeting other receptor tyrosine kinases with antibodies.     

Tamoxifen inhibits tumor cell invasion and metastasis in mouse melanoma through suppression of PKC/MEK/ERK and PKC/PI3K/Akt pathways

In melanoma, several signaling pathways are constitutively activated. Among these, the protein kinase C (PKC) signaling pathways are activated through multiple signal transduction molecules and appear to play major roles in melanoma progression. Recently, it has been reported that tamoxifen, an anti-estrogen reagent, inhibits PKC signaling in estrogen-negative and estrogen-independent cancer cell lines. Thus, we investigated whether tamoxifen inhibited tumor cell invasion and metastasis in mouse melanoma cell line B16BL6. Tamoxifen significantly inhibited lung metastasis, cell migration, and invasion at concentrations that did not show anti-proliferative effects on B16BL6 cells. Tamoxifen also inhibited the mRNA expressions and protein activities of matrix metalloproteinases (MMPs). Furthermore, tamoxifen suppressed phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt through the inhibition of PKCα and PKCδ phosphorylation. However, other signal transduction factor, such as p38 mitogen-activated protein kinase (p38MAPK) was unaffected. The results indicate that tamoxifen suppresses the PKC/mitogen-activated protein kinase kinase (MEK)/ERK and PKC/phosphatidylinositol-3 kinase (PI3K)/Akt pathways, thereby inhibiting B16BL6 cell migration, invasion, and metastasis. Moreover, tamoxifen markedly inhibited not only developing but also clinically evident metastasis. These findings suggest that tamoxifen has potential clinical applications for the treatment of tumor cell metastasis.     

Fibroblast growth factor signaling controlling bone formation: An update

Fibroblast Growth Factors (FGFs) regulate prenatal and postnatal bone formation through activation of FGF receptors (FGFR) and downstream signaling events. During the last decade, major advances have been made in our understanding of the mechanisms by which FGF/FGFR signaling controls osteoprogenitor cell replication and osteoblast differentiation and function. The analysis of the phenotype induced by FGF invalidation and mutations in FGFR allowed to delineate key FGF signaling pathways that regulate osteoblastogenesis. Molecular genomic studies led to identify target genes that are controlled by FGF/FGFR signaling and govern osteoblasts. The analysis of intracellular signaling pathways showed the importance of functional crosstalks between FGF signaling and other pathways in the regulation of bone formation. These recent progresses in the mechanisms underlying FGF/FGFR signaling may provide a molecular basis for developing therapeutic strategies in human skeletal dysplasias.     

Using AI-Based Evolutionary Algorithms to Elucidate Adult Brain Tumor (Glioma) Etiology Associated with IDH1 for Therapeutic Target Identification

Adult brain tumors (glioma) represent a cancer of unmet need where standard-of-care is non-curative; thus, new therapies are urgently needed. It is unclear whether isocitrate dehydrogenases (IDH1/2) when not mutated have any role in gliomagenesis or tumor growth. Nevertheless, IDH1 is overexpressed in glioblastoma (GBM), which could impact upon cellular metabolism and epigenetic reprogramming. This study characterizes IDH1 expression and associated genes and pathways. A novel biomarker discovery pipeline using artificial intelligence (evolutionary algorithms) was employed to analyze IDH-wildtype adult gliomas from the TCGA LGG-GBM cohort. Ninety genes whose expression correlated with IDH1 expression were identified from: (1) All gliomas, (2) primary GBM, and (3) recurrent GBM tumors. Genes were overrepresented in ubiquitin-mediated proteolysis, focal adhesion, mTOR signaling, and pyruvate metabolism pathways. Other non-enriched pathways included O-glycan biosynthesis, notch signaling, and signaling regulating stem cell pluripotency (PCGF3). Potential prognostic (TSPYL2, JAKMIP1, CIT, TMTC1) and two diagnostic (MINK1, PLEKHM3) biomarkers were downregulated in GBM. Their gene expression and methylation were negatively and positively correlated with IDH1 expression, respectively. Two diagnostic biomarkers (BZW1, RCF2) showed the opposite trend. Prognostic genes were not impacted by high frequencies of molecular alterations and only one (TMTC1) could be validated in another cohort. Genes with mechanistic links to IDH1 were involved in brain neuronal development, cell proliferation, cytokinesis, and O-mannosylation as well as tumor suppression and anaplerosis. Results highlight metabolic vulnerabilities and therapeutic targets for use in future clinical trials.