Oncometabolite induced primary cilia loss in pheochromocytoma

Primary cilia are sensory organelles involved in regulation of cellular signaling. Cilia loss is frequently observed in tumors; yet, the responsible mechanisms and consequences for tumorigenesis remain unclear. We demonstrate that cilia structure and function is disrupted in human pheochromocytomas – endocrine tumors of the adrenal medulla. This is concomitant with transcriptional changes within cilia-mediated signaling pathways that are associated with tumorigenesis generally and pheochromocytomas specifically. Importantly, cilia loss was most dramatic in patients with germline mutations in the pseudohypoxia-linked genes SDHx and VHL. Using a pheochromocytoma cell line derived from rat, we show that hypoxia and oncometabolite-induced pseudohypoxia are key drivers of cilia loss and identify that this is dependent on activation of an Aurora-A/HDAC6 cilia resorption pathway. We also show cilia loss drives dramatic transcriptional changes associated with proliferation and tumorigenesis. Our data provide evidence for primary cilia dysfunction contributing to pathogenesis of pheochromocytoma by a hypoxic/pseudohypoxic mechanism and implicates oncometabolites as ciliary regulators. This is important as pheochromocytomas can cause mortality by mechanisms including catecholamine production and malignant transformation, while hypoxia is a general feature of solid tumors. Moreover, pseudohypoxia-induced cilia resorption can be pharmacologically inhibited, suggesting potential for therapeutic intervention

Modification of splicing with antisense oligonucleotides in the insulin receptor exon 11 and apolipoprotein B exon 26

Background - The alternatively spliced insulin receptor (IR) exon 11 (36 nucleotides) and the constitutively spliced Apolipoprotein B (APOB) exon 26 (7572 nucleotides) are examples of the shortest and longest exons in the genome. Aim - The aim of this study was to investigate the regulation of splicing of these 2 exons in cell culture using 2'-O-methyl RNA antisense oligonucleotides (ASOs) and peptide nucleic acid (PNA)-peptide hybrid ASOs. Methods - ASOs were designed to target key sequences involved in the splicing of the IR exon 11 and exonic splicing silencer sequences (ESS) in APOB exon 26. HepG2 cells were reverse-transfected with the ASOs for 48 hours, mRNA harvested and RT-PCR was performed to amplify the IR isoform and APOB cDNAs which were separated by PAGE and quantified. Results Insulin receptor exon 11 - 2'-O-methyl RNA ASOs targeted to two intronic sites, the 3' half of exon 11 and spanning the entire exon caused significant exon skipping. PNA-peptide hybrids predicted to increase exon 11 splicing, paradoxically caused exon skipping. PNA-peptide hybrids with 3' tails caused exon 11 skipping more effectively than hybrids with 5' tails. Apolipoprotein B exon 26 - Only combinations of 2'-O-methyl RNA ASOs targeting multiple ESSs in APOB exon 26 caused a small proportion of aberrant 3 1 1 ! ..l I splicing. This consisted of complete exon 26 skipping and the selection of a downstream cryptic 3' splice site in preference to the native 3' splice site. Discussion - Exclusion of the IR exon 11 can be induced by targeting a combination of intronic or exonic sequences. PNA-peptide hybrid ASOs were unable to increase exon 11 splicing. The aberrant splicing of large constitutive exons such as APOB exon 26 can be induced by targeting multiple ESS sites along its course.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Cortical gyrification in women and men and the (missing) link to prenatal androgens.

Publication status: PublishedPrevious studies have reported sex differences in cortical gyrification. Since most cortical folding is principally defined in utero, sex chromosomes as well as gonadal hormones are likely to influence sex-specific aspects of local gyrification. Classic congenital adrenal hyperplasia (CAH) causes high levels of androgens during gestation in females, whereas levels in males are largely within the typical male range. Therefore, CAH provides an opportunity to study the possible effects of prenatal androgens on cortical gyrification. Here, we examined the vertex-wise absolute mean curvature-a common estimate for cortical gyrification-in individuals with CAH (33 women and 20 men) and pair-wise matched controls (33 women and 20 men). There was no significant main effect of CAH and no significant CAH-by-sex interaction. However, there was a significant main effect of sex in five cortical regions, where gyrification was increased in women compared to men. These regions were located on the lateral surface of the brain, specifically left middle frontal (rostral and caudal), right inferior frontal, left inferior parietal, and right occipital. There was no cortical region where gyrification was increased in men compared to women. Our findings do not only confirm prior reports of increased cortical gyrification in female brains but also suggest that cortical gyrification is not significantly affected by prenatal androgen exposure. Instead, cortical gyrification might be determined by sex chromosomes either directly or indirectly-the latter potentially by affecting the underlying architecture of the cortex or the size of the intracranial cavity, which is smaller in women

Cortical gyrification in women and men and the (missing) link to prenatal androgens

Previous studies have reported sex differences in cortical gyrification. Since most cortical folding is principally defined in utero, sex chromosomes as well as gonadal hormones are likely to influence sex-specific aspects of local gyrification. Classic congenital adrenal hyperplasia (CAH) causes high levels of androgens during gestation in females, whereas levels in males are largely within the typical male range. Therefore, CAH provides an opportunity to study the possible effects of prenatal androgens on cortical gyrification. Here, we examined the vertex-wise absolute mean curvature—a common estimate for cortical gyrification—in individuals with CAH (33 women and 20 men) and pair-wise matched controls (33 women and 20 men). There was no significant main effect of CAH and no significant CAH-by-sex interaction. However, there was a significant main effect of sex in five cortical regions, where gyrification was increased in women compared to men. These regions were located on the lateral surface of the brain, specifically left middle frontal (rostral and caudal), right inferior frontal, left inferior parietal, and right occipital. There was no cortical region where gyrification was increased in men compared to women. Our findings do not only confirm prior reports of increased cortical gyrification in female brains but also suggest that cortical gyrification is not significantly affected by prenatal androgen exposure. Instead, cortical gyrification might be determined by sex chromosomes either directly or indirectly—the latter potentially by affecting the underlying architecture of the cortex or the size of the intracranial cavity, which is smaller in women