VEGF receptor 2/-3 heterodimers detected in situ by proximity ligation on angiogenic sprouts

Peer reviewe

Post-translational regulation of Myc oncoprotein function

The c-Myc proto-oncogene encodes a transcription factor that controls genes involved in cell cycle regulation, cell growth, apoptosis and other cellular processes together with its partner Max. c-Myc has been shown to be altered in a wide variety of human tumors. This thesis focuses on post-translational regulation of Myc function. The aims was firstly to elucidate mechanisms of regulation of Myc degradation and components involved, and secondly to understand the mechanism(s) behind IFN-γ-induced inactivation of Myc. The c-Myc oncoprotein is short–lived phosphoprotein. We have found that Myc is tightly regulated via ubiquitin/proteasome-mediated turnover and that mutation of the Thr-58 phosphorylation site, which is frequently mutated in both in Burkitt’s lymphoma leads to stabilization of c-Myc. We have further established that the E3-ubiquitin ligase SCFSkp2 associates with c-Myc in late G1 and S-phase of the cell cycle, inducing not only ubiquitin-mediated degradation of c-Myc but surprisingly transcriptional activation of c-Myc target genes. Skp2 as well as proteasomal subunits were shown to be recruited to a Myc target promoter in vivo in a Myc-dependent manner. The second part of the thesis shows that the cytokine IFN-γ restore differentiation and cell cycle arrest in v-Myc expressing monocytic cells through inhibition of Myc-induced transcription, Myc DNA-binding and destabilization of Myc:Max heterodimers, correlating with dephosphorylation of Myc. We further found that IFN-γ reduces phosphorylation of Ser-62 and increases ubiquitin/proteasome-mediated degradation of Myc in a Ser-62 dependent manner.CycE/CDK2 was identified as a Ser-62 kinase in vivo and in vitro. IFN-γ-induced degradation and Ser-62 dephosphorylation correlated with inactivation of CDK2 through p27Kip1, which was shown to be required for this process. In conclusion, the thesis suggests that ubiquitin/proteasome-mediated turnover is an essential level of regulation of Myc function that may have important future implications for treatment of tumors with deregulated Myc expression

Combined IFN-gamma and retinoic acid treatment targets the N-Myc/Max/Mad1 network resulting in repression of N-Myc target genes in MYCN-amplified neuroblastoma cells

The MYCN protooncogene is involved in the control of cell proliferation, differentiation, and survival of neuroblasts. Deregulation of MYCN by gene amplification contributes to neuroblastoma development and is strongly correlated to advanced disease and poor outcome, emphasizing the urge for new therapeutic strategies targeting MYCN function. The transcription factor N-Myc, encoded by MYCN, regulates numerous genes together with its partner Max, which also functions as a cofactor for the Mad/Mnt family of Myc antagonists/transcriptional repressors. We and others have previously reported that IFN-gamma synergistically potentiates retinoic acid (RA)induced sympathetic differentiation and growth inhibition in neuroblastoma cells. This study shows that combined treatment of MYCN-amplified neuroblastorna cells with RA+IFN-gamma down-regulates N-Myc protein expression through increased protein turnover, up-regulates Mad1 mRNA and protein, and reduces N-Myc/Max heteroclimerization. This results in a shift of occupancy at the ornithine decarboxylase N-Myc/Mad1 target promoter in vivo from N-Myc/Max to Madl/Max predominance, correlating with histone H4 deacetylation, indicative of a chromatin structure typical of a transcriptionally repressed state. This is further supported by data showing that RA + IFN-gamma treatment strongly represses expression of N-Myc/Mad1 target genes ornithine decarboxylase and hTERT. Our results suggest that combined IFN-gamma and RA signaling can form a basis for new therapeutic strategies targeting N-Myc function for patients with high-risk, MYCN-amplified neuroblastoma

Interferon-γ-induced p27KIP1 binds to and targets MYC for proteasome-mediated degradation

The Myc oncoprotein is tightly regulated at multiple levels including ubiquitin-mediated protein turnover. We recently demonstrated that inhibition of Cdk2-mediated phosphorylation of Myc at Ser-62 pharmacologically or through interferon (IFN)-γ-induced expression of p27Kip1 (p27) repressed Myc's activity to suppress cellular senescence and differentiation. In this study we identified an additional activity of p27 to interfere with Myc independent of Ser-62 phosphorylation. p27 is required and sufficient for IFN-γ-induced turnover of Myc. p27 interacted with Myc in the nucleus involving the C-termini of the two proteins, including Myc box 4 of Myc. The C-terminus but not the Cdk2 binding fragment of p27 was sufficient for inducing Myc degradation. Protein expression data of The Cancer Genome Atlas breast invasive carcinoma set revealed significantly lower Myc protein levels in tumors with highly expressed p27 lacking phosphorylation at Thr-157 - a marker for active p27 localized in the nucleus. Further, these conditions correlated with favorable tumor stage and patient outcome. This novel regulation of Myc by IFN-γ/p27KIP1 potentially offers new possibilities for therapeutic intervention in tumors with deregulated Myc

Maternal stress, early life factors and infant salivary cortisol levels

Background: Salivary cortisol (SC), a commonly used biomarker for stress, may be disrupted by negative events in pregnancy, at birth and in infancy. We aimed to explore if maternal perceived stress (PSS) in or after pregnancy and SC levels in pregnancy were associated with SC in early infancy, and, secondly, to identify early life factors associated with infants’ SC levels (iSC). Methods: At 3 months of age, SC was analyzed in 1057 infants participating in a Nordic prospective mother-child birth cohort study. Maternal PSS was available from questionnaires at 18- and 34-week gestational age (GA) and 3-month post-partum, and SC was analyzed at 18-week GA. Early life factors included sociodemographic and infant feeding from questionnaires, and birth data from medical charts. Associations to iSC were analyzed by Spearman correlation and multinomial logistic regression analyses. Results: In this exploratory study neither PSS at any time point nor maternal SC (mSC) were associated with iSC. Higher birth weight was associated with higher levels of iSC, while inverse associations were observed in infants to a mother not living with a partner and mixed bottle/breastfeeding. Conclusions: Maternal stress was not associated with iSC levels, while birth weight, single motherhood and infant feeding may influence iSC levels

Maternal Stress, Early Life Factors and Infant Salivary Cortisol Levels

Background: Salivary cortisol (SC), a commonly used biomarker for stress, may be disrupted by negative events in pregnancy, at birth and in infancy. We aimed to explore if maternal perceived stress (PSS) in or after pregnancy and SC levels in pregnancy were associated with SC in early infancy, and, secondly, to identify early life factors associated with infants’ SC levels (iSC). Methods: At 3 months of age, SC was analyzed in 1057 infants participating in a Nordic prospective mother-child birth cohort study. Maternal PSS was available from questionnaires at 18- and 34-week gestational age (GA) and 3-month post-partum, and SC was analyzed at 18-week GA. Early life factors included sociodemographic and infant feeding from questionnaires, and birth data from medical charts. Associations to iSC were analyzed by Spearman correlation and multinomial logistic regression analyses. Results: In this exploratory study neither PSS at any time point nor maternal SC (mSC) were associated with iSC. Higher birth weight was associated with higher levels of iSC, while inverse associations were observed in infants to a mother not living with a partner and mixed bottle/breastfeeding. Conclusions: Maternal stress was not associated with iSC levels, while birth weight, single motherhood and infant feeding may influence iSC levels