Transcriptomic analysis of dystrophin RNAi knockdown reveals a central role for dystrophin in muscle differentiation and contractile apparatus organization

<p>Abstract</p> <p>Background</p> <p>Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disorder caused by mutations in the dystrophin gene. DMD has a complex and as yet incompletely defined molecular pathophysiology hindering development of effective ameliorative approaches. Transcriptomic studies so far conducted on dystrophic cells and tissues suffer from non-specific changes and background noise due to heterogeneous comparisons and secondary pathologies. A study design in which a perfectly matched control cell population is used as reference for transcriptomic studies will give a much more specific insight into the effects of dystrophin deficiency and DMD pathophysiology.</p> <p>Results</p> <p>Using RNA interference (RNAi) to knock down dystrophin in myotubes from C57BL10 mice, we created a homogenous model to study the transcriptome of dystrophin-deficient myotubes. We noted significant differences in the global gene expression pattern between these myotubes and their matched control cultures. In particular, categorical analyses of the dysregulated genes demonstrated significant enrichment of molecules associated with the components of muscle cell contractile unit, ion channels, metabolic pathways and kinases. Additionally, some of the dysregulated genes could potentially explain conditions and endophenotypes associated with dystrophin deficiency, such as dysregulation of calcium homeostasis (<it>Pvalb </it>and <it>Casq1</it>), or cardiomyopathy (<it>Obscurin</it>, <it>Tcap</it>). In addition to be validated by qPCR, our data gains another level of validity by affirmatively reproducing several independent studies conducted previously at genes and/or protein levels <it>in vivo </it>and <it>in vitro</it>.</p> <p>Conclusion</p> <p>Our results suggest that in striated muscles, dystrophin is involved in orchestrating proper development and organization of myofibers as contractile units, depicting a novel pathophysiology for DMD where the absence of dystrophin results in maldeveloped myofibers prone to physical stress and damage. Therefore, it becomes apparent that any gene therapy approaches for DMD should target early stages in muscle development to attain a maximum clinical benefit. With a clear and specific definition of the transcriptome of dystrophin deficiency, manipulation of identified dysregulated molecules downstream of dystrophin may lead to novel ameliorative approaches for DMD.</p

Human PTCHD3 nulls: rare copy number and sequence variants suggest a non-essential gene

<p>Abstract</p> <p>Background</p> <p>Copy number variations (CNVs) can contribute to variable degrees of fitness and/or disease predisposition. Recent studies show that at least 1% of any given genome is copy number variable when compared to the human reference sequence assembly. Homozygous deletions (or CNV nulls) that are found in the normal population are of particular interest because they may serve to define non-essential genes in human biology.</p> <p>Results</p> <p>In a genomic screen investigating CNV in Autism Spectrum Disorders (ASDs) we detected a heterozygous deletion on chromosome 10p12.1, spanning the Patched-domain containing 3 (<it>PTCHD3</it>) gene, at a frequency of ~1.4% (6/427). This finding seemed interesting, given recent discoveries on the role of another Patched-domain containing gene (<it>PTCHD1</it>) in ASD. Screening of another 177 ASD probands yielded two additional heterozygous deletions bringing the frequency to 1.3% (8/604). The deletion was found at a frequency of ~0.73% (27/3,695) in combined control population from North America and Northern Europe predominately of European ancestry. Screening of the human genome diversity panel (HGDP-CEPH) covering worldwide populations yielded deletions in 7/1,043 unrelated individuals and those detected were confined to individuals of European/Mediterranean/Middle Eastern ancestry. Breakpoint mapping yielded an identical 102,624 bp deletion in all cases and controls tested, suggesting a common ancestral event. Interestingly, this CNV occurs at a break of synteny between humans and mouse. Considering all data, however, no significant association of these rare <it>PTCHD3 </it>deletions with ASD was observed. Notwithstanding, our RNA expression studies detected <it>PTCHD3 </it>in several tissues, and a novel shorter isoform for <it>PTCHD3 </it>was characterized. Expression in transfected COS-7 cells showed <it>PTCHD3 </it>isoforms colocalize with calnexin in the endoplasmic reticulum. The presence of a patched (Ptc) domain suggested a role for <it>PTCHD3 </it>in various biological processes mediated through the Hedgehog (Hh) signaling pathway. However, further investigation yielded one individual harboring a homozygous deletion (<it>PTCHD3 </it>null) without ASD or any other overt abnormal phenotype. Exon sequencing of <it>PTCHD3 </it>in other individuals with deletions revealed compound point mutations also resulting in a null state.</p> <p>Conclusion</p> <p>Our data suggests that <it>PTCHD3 </it>may be a non-essential gene in some humans and characterization of this novel CNV at 10p12.1 will facilitate population and disease studies.</p

Image_2_Immunotherapy with STING and TLR9 agonists promotes synergistic therapeutic efficacy with suppressed cancer-associated fibroblasts in colon carcinoma.jpg

The innate immune sensing of nucleic acids using effective immunoadjuvants is critical for increasing protective immune responses against cancer. Stimulators of interferon genes (STING) and toll-like receptor 9 (TLR9) agonists are considered promising candidates in several preclinical tumor models with the potential to be used in clinical settings. However, the effects of such treatment on tumor stroma are currently unknown. In this study, we investigated the immunotherapeutic effects of ADU-S100 as a STING agonist and CpG ODN1826 as a TLR9 agonist in a preclinical model of colon carcinoma. Tumor-bearing mice were treated intratumorally on days 10 and 16 post-tumor inoculation with ADU-S100 and CpG ODN1826. Cytokine profiles in the tumor and spleen, tumor cell apoptosis, the infiltration of immune cells, and cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME) were evaluated to identify the immunological mechanisms after treatment. The powerful antitumor activity of single and combination treatments, the upregulation of the expression of pro-inflammatory cytokines in the tumor and spleen, and the recruitment and infiltration of the TME by immune cells revealed the synergism of immunoadjuvants in the eradication of the colon carcinoma model. Remarkably, the significant downregulation of CAFs in the TME indicated that suppression of tumorigenesis occurred after immunoadjuvant therapy. The results illustrate the potential of targeting the STING and TLR9 pathways as powerful immunoadjuvants in the treatment of preclinical colon carcinoma and the possibility of harnessing these pathways in future therapeutic approaches.</p

Image_3_Immunotherapy with STING and TLR9 agonists promotes synergistic therapeutic efficacy with suppressed cancer-associated fibroblasts in colon carcinoma.jpg

The innate immune sensing of nucleic acids using effective immunoadjuvants is critical for increasing protective immune responses against cancer. Stimulators of interferon genes (STING) and toll-like receptor 9 (TLR9) agonists are considered promising candidates in several preclinical tumor models with the potential to be used in clinical settings. However, the effects of such treatment on tumor stroma are currently unknown. In this study, we investigated the immunotherapeutic effects of ADU-S100 as a STING agonist and CpG ODN1826 as a TLR9 agonist in a preclinical model of colon carcinoma. Tumor-bearing mice were treated intratumorally on days 10 and 16 post-tumor inoculation with ADU-S100 and CpG ODN1826. Cytokine profiles in the tumor and spleen, tumor cell apoptosis, the infiltration of immune cells, and cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME) were evaluated to identify the immunological mechanisms after treatment. The powerful antitumor activity of single and combination treatments, the upregulation of the expression of pro-inflammatory cytokines in the tumor and spleen, and the recruitment and infiltration of the TME by immune cells revealed the synergism of immunoadjuvants in the eradication of the colon carcinoma model. Remarkably, the significant downregulation of CAFs in the TME indicated that suppression of tumorigenesis occurred after immunoadjuvant therapy. The results illustrate the potential of targeting the STING and TLR9 pathways as powerful immunoadjuvants in the treatment of preclinical colon carcinoma and the possibility of harnessing these pathways in future therapeutic approaches.</p

Image_4_Immunotherapy with STING and TLR9 agonists promotes synergistic therapeutic efficacy with suppressed cancer-associated fibroblasts in colon carcinoma.jpg

The innate immune sensing of nucleic acids using effective immunoadjuvants is critical for increasing protective immune responses against cancer. Stimulators of interferon genes (STING) and toll-like receptor 9 (TLR9) agonists are considered promising candidates in several preclinical tumor models with the potential to be used in clinical settings. However, the effects of such treatment on tumor stroma are currently unknown. In this study, we investigated the immunotherapeutic effects of ADU-S100 as a STING agonist and CpG ODN1826 as a TLR9 agonist in a preclinical model of colon carcinoma. Tumor-bearing mice were treated intratumorally on days 10 and 16 post-tumor inoculation with ADU-S100 and CpG ODN1826. Cytokine profiles in the tumor and spleen, tumor cell apoptosis, the infiltration of immune cells, and cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME) were evaluated to identify the immunological mechanisms after treatment. The powerful antitumor activity of single and combination treatments, the upregulation of the expression of pro-inflammatory cytokines in the tumor and spleen, and the recruitment and infiltration of the TME by immune cells revealed the synergism of immunoadjuvants in the eradication of the colon carcinoma model. Remarkably, the significant downregulation of CAFs in the TME indicated that suppression of tumorigenesis occurred after immunoadjuvant therapy. The results illustrate the potential of targeting the STING and TLR9 pathways as powerful immunoadjuvants in the treatment of preclinical colon carcinoma and the possibility of harnessing these pathways in future therapeutic approaches.</p

Image_1_Immunotherapy with STING and TLR9 agonists promotes synergistic therapeutic efficacy with suppressed cancer-associated fibroblasts in colon carcinoma.jpg

The innate immune sensing of nucleic acids using effective immunoadjuvants is critical for increasing protective immune responses against cancer. Stimulators of interferon genes (STING) and toll-like receptor 9 (TLR9) agonists are considered promising candidates in several preclinical tumor models with the potential to be used in clinical settings. However, the effects of such treatment on tumor stroma are currently unknown. In this study, we investigated the immunotherapeutic effects of ADU-S100 as a STING agonist and CpG ODN1826 as a TLR9 agonist in a preclinical model of colon carcinoma. Tumor-bearing mice were treated intratumorally on days 10 and 16 post-tumor inoculation with ADU-S100 and CpG ODN1826. Cytokine profiles in the tumor and spleen, tumor cell apoptosis, the infiltration of immune cells, and cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME) were evaluated to identify the immunological mechanisms after treatment. The powerful antitumor activity of single and combination treatments, the upregulation of the expression of pro-inflammatory cytokines in the tumor and spleen, and the recruitment and infiltration of the TME by immune cells revealed the synergism of immunoadjuvants in the eradication of the colon carcinoma model. Remarkably, the significant downregulation of CAFs in the TME indicated that suppression of tumorigenesis occurred after immunoadjuvant therapy. The results illustrate the potential of targeting the STING and TLR9 pathways as powerful immunoadjuvants in the treatment of preclinical colon carcinoma and the possibility of harnessing these pathways in future therapeutic approaches.</p