Involvement of abnormal dystroglycan expression and matriglycan levels in cancer pathogenesis.

Dystroglycan (DG) is a glycoprotein composed of two subunits that remain non-covalently bound at the plasma membrane: α-DG, which is extracellular and heavily O-mannosyl glycosylated, and ß-DG, an integral transmembrane polypeptide. α-DG is involved in the maintenance of tissue integrity and function in the adult, providing an O-glycosylation-dependent link for cells to their extracellular matrix. ß-DG in turn contacts the cytoskeleton via dystrophin and participates in a variety of pathways transmitting extracellular signals to the nucleus. Increasing evidence exists of a pivotal role of DG in the modulation of normal cellular proliferation. In this context, deficiencies in DG glycosylation levels, in particular those affecting the so-called matriglycan structure, have been found in an ample variety of human tumors and cancer-derived cell lines. This occurs together with an underexpression of the DAG1 mRNA and/or its α-DG (core) polypeptide product or, more frequently, with a downregulation of ß-DG protein levels. These changes are in general accompanied in tumor cells by a low expression of genes involved in the last steps of the α-DG O-mannosyl glycosylation pathway, namely POMT1/2, POMGNT2, CRPPA, B4GAT1 and LARGE1/2. On the other hand, a series of other genes acting earlier in this pathway are overexpressed in tumor cells, namely DOLK, DPM1/2/3, POMGNT1, B3GALNT2, POMK and FKTN, hence exerting instead a pro-oncogenic role. Finally, downregulation of ß-DG, altered ß-DG processing and/or impaired ß-DG nuclear levels are increasingly found in human tumors and cell lines. It follows that DG itself, particular genes/proteins involved in its glycosylation and/or their interactors in the cell could be useful as biomarkers of certain types of human cancer, and/or as molecular targets of new therapies addressing these neoplasms.

Retinal Proteomics of a Mouse Model of Dystroglycanopathies Reveals Molecular Alterations in Photoreceptors.

Mutations in the POMT1 gene, encoding a protein O-mannosyltransferase essential for α-dystroglycan (α-DG) glycosylation, are frequently observed in a group of rare congenital muscular dystrophies, collectively known as dystroglycanopathies. However, it is hitherto unclear whether the effects seen in affected patients can be fully ascribed to α-DG hypoglycosylation. To study this, here we used comparative mass spectrometry-based proteomics and immunofluorescence microscopy and investigated the changes in the retina of mice in which Pomt1 is specifically knocked out in photoreceptor cells. Our results demonstrate significant proteomic changes and associated structural alteration in photoreceptor cells of Pomt1 cKO mice. In addition to the effects related to impaired α-DG O-mannosylation, we observed morphological alterations in the outer segment that are associated with dysregulation of a relatively understudied POMT1 substrate (KIAA1549), BBSome proteins, and retinal stress markers. In conclusion, our study provides new hypotheses to explain the phenotypic changes that are observed in the retina of patients with dystroglycanopathies.

Correction: Radiotherapy resistance acquisition in Glioblastoma. Role of SOCS1 and SOCS3.

[This corrects the article DOI: 10.1371/journal.pone.0212581.].

Radiotherapy resistance acquisition in Glioblastoma. Role of SOCS1 and SOCS3.

Glioblastoma multiforme (GBM) is a poor prognosis type of tumour due to its resistance to chemo and radiotherapy. SOCS1 and SOCS3 have been associated with tumour progression and response to treatments in different kinds of cancers, including GBM. In this study, cell lines of IDH-wildtype GBM from primary cultures were obtained, and the role of SOCS1 and SOCS3 in the radiotherapy response was analysed. Fifty-two brain aspirates from GBM patients were processed, and six new cell lines of IDH-wildtype GBM were established. These new cell lines were characterized according to the WHO classification of CNS tumours. SOCS1 and SOCS3 expression levels were determined, at mRNA level by Q-PCR, at protein level by immunocytochemistry, and Western blot analysis. The results showed that SOCS1 and SOCS3 are overexpressed in GBM, as compared to a non-tumoral brain RNA pool. SOCS1 and SOCS3 expression were reduced by siRNA, and it was found that SOCS3 inhibition increases radioresistance in GBM cell lines, suggesting a key role of SOCS3 in radioresistant acquisition. In addition, radioresistant clonal populations obtained by selective pressure on these cell cultures also showed a significant decrease in SOCS3 expression, while SOCS1 remained unchanged. Furthermore, the induction of SOCS3 expression, under a heterologous promoter, in a radiotherapy resistant GBM cell line increased its radiosensitivity, supporting an important implication of SOCS3 in radiotherapy resistance acquisition. Finally, the treatment with TSA in the most radioresistant established cell line produced an increase in the effect of radiotherapy, that correlated with an increase in the expression of SOCS3. These effects of TSA disappeared if the increase in the expression of SOCS3 prevented with an siRNA against SOCS3. Thus, SOCS3 signal transduction pathway (JAK/STAT) could be useful to unmask new putative targets to improve radiotherapy response in GBM.

Impairment of photoreceptor ribbon synapses in a novel Pomt1 conditional knockout mouse model of dystroglycanopathy.

Hypoglycosylation of α-dystroglycan (α-DG) resulting from deficiency of protein O-mannosyltransferase 1 (POMT1) may cause severe neuromuscular dystrophies with brain and eye anomalies, named dystroglycanopathies. The retinal involvement of these disorders motivated us to generate a conditional knockout (cKO) mouse experiencing a Pomt1 intragenic deletion (exons 3-4) during the development of photoreceptors, mediated by the Cre recombinase expressed from the cone-rod homeobox (Crx) gene promoter. In this mouse, retinal α-DG was unglycosylated and incapable of binding laminin. Retinal POMT1 deficiency caused significant impairments in both electroretinographic recordings and optokinetic reflex in Pomt1 cKO mice, and immunohistochemical analyses revealed the absence of ß-DG and of the α-DG-interacting protein, pikachurin, in the outer plexiform layer (OPL). At the ultrastructural level, noticeable alterations were observed in the ribbon synapses established between photoreceptors and bipolar cells. Therefore, O-mannosylation of α-DG in the retina carried out by POMT1 is crucial for the establishment of proper synapses at the OPL and transmission of visual information from cones and rods to their postsynaptic neurons.

Expression in retinal neurons of fukutin and FKRP, the protein products of two dystroglycanopathy-causative genes.

Purpose: Dystroglycanopathies are a heterogeneous group of recessive neuromuscular dystrophies that affect the muscle, brain and retina, and are caused by deficiencies in the O-glycosylation of α-dystroglycan. This post-translational modification is essential for the formation and maintenance of ribbon synapses in the retina. Fukutin and fukutin-related protein (FKRP) are two glycosyltransferases whose deficiency is associated with severe dystroglycanopathies. These enzymes carry out in vitro the addition of a tandem ribitol 5-phosphate moiety to the so-called core M3 phosphotrisaccharide of α-dystroglycan. However, their expression pattern and function in the healthy mammalian retina has not so far been investigated. In this work, we have addressed the expression of the FKTN (fukutin) and FKRP genes in the retina of mammals, and characterized the distribution pattern of their protein products in the adult mouse retina and the 661W photoreceptor cell line. Methods: By means of reverse transcription (RT)-PCR and immunoblotting, we have studied the expression at the mRNA and protein levels of the fukutin and FKRP genes in different mammalian species, from rodents to humans. Immunofluorescence confocal microscopy analyses were performed to characterize the distribution profile of their protein products in mouse retinal sections and in 661W cultured cells. Results: Both genes were expressed at the mRNA and protein levels in the neural retina of all mammals studied. Fukutin was present in the cytoplasmic and nuclear fractions in the mouse retina and 661W cells, and accumulated in the endoplasmic reticulum. FKRP was located in the cytoplasmic fraction in the mouse retina and concentrated in the Golgi complex. However, and in contrast to retinal tissue, FKRP additionally accumulated in the nucleus of the 661W photoreceptors. Conclusions: Our results suggest that fukutin and FKRP not only participate in the synthesis of O-mannosyl glycans added to α-dystroglycan in the endoplasmic reticulum and Golgi complex, but that they could also play a role, that remains to be established, in the nucleus of retinal neurons.