Response: Anxiety and behaviour in and beyond ASD; does the idea of 'PDA' really help? - a response to Green (2020).

We acknowledge Green's viewpoint and agree that currently the literature does not support the validity of PDA as an independent syndrome. However, the controversy is real and exists for a reason. We therefore argue that it is important to move beyond labelling and the potentially unhelpful implications of discussion around whether or not it is a condition, diagnosis or a co-morbid condition, to a point of recognition of the phenomenon. We argue that what we need is more accurate description of these behaviours and better measurement to allow us to answer key questions about this phenomenon, whatever we name it. In our paper (Stuart et al, Child and Adolescent Mental Health, 2019) and response, we are trying to move the debate on from opinion and anecdote to be more data-driven, which hopefully leads to increased identification of what research needs to be done. We build on this argument by outlining our second proposed and yet to be published paper exploring the role that IU has in the relationship between ASD, PDA (however we label this) and anxiety.

Intolerance of uncertainty and anxiety as explanatory frameworks for extreme demand avoidance in children and adolescents.

BACKGROUND: Pathological demand avoidance (PDA) is a proposed subtype of autism spectrum disorder (ASD), characterised by extreme avoidance of demands. Demand avoidant behaviour has been proposed to be driven by an anxious need to be in control, although has never been explicitly studied. Emerging evidence suggests intolerance of uncertainty (IU) and anxiety may explain the behaviours seen in ASD. We propose these concepts may be useful starting points for furthering understanding of PDA. METHODS: In Study 1, quantitative methods examined the relationship between PDA, IU and anxiety using data collected in an online survey (N = 214). The sample included cases with clinically diagnosed PDA (n = 69) and those with no clinical diagnosis but parent-identified features of PDA (n = 151). 'Children with a diagnosis of PDA scored significantly higher on the IUS-P (t(212) = 2.45, p < .05) compared to those without a diagnosis of PDA. PDA diagnosis did not impact on scores on any other measure.' In Study 2, a selection of Study 1 participants (n = 11) were followed up with a telephone interview to gain descriptive data relating to PDA and its association with IU and anxiety. RESULTS: Regression analyses indicate that demand avoidant behaviour can be conceptualised in part as a possible attempt to increase certainty and predictability to alleviate increasing anxiety. Children and young people with PDA employed varying strategies to manage IU depending on the level of demand presented and degree of anxiety generated. These strategies can be represented by different features of the behaviour profile seen in PDA (control behaviour, withdrawal to fantasy, and meltdown). These behavioural features of PDA showed differential relationships with IU and anxiety, although all were predicted by IU, only meltdown demonstrated a mediation effect by anxiety. CONCLUSIONS: This study represents one of the first attempts to conceptualise and understand the behavioural features of the PDA profile in children and young people. It builds upon emerging evidence from the ASD literature that IU is a relevant construct for conceptualising demand avoidant behaviour in children who show PDA behaviour. This has potential clinical implications for the assessment and management of PDA in children and young people.

Response: Demand Avoidance Phenomena: a manifold issue? Intolerance of uncertainty and anxiety as explanatory frameworks for extreme demand avoidance in children and adolescents - a response to Woods (2020).

This paper is in response to the commentary written by Richard Woods in which he attempts to provide support for his Monotropism autism theory and the research indicating that Demand Avoidance Phenomena may not be developmentally persistent (Woods, 2019). We acknowledge the continuing controversy around the proposed construct of PDA and the clinical dilemma faced by professionals, within the United Kingdom, following increased demand from families seeking assessment and support. We appreciate that research on this topic is scarce and understanding of PDA behaviours remains limited and that methodological improvements are required. However, it is important to remember that anxiety, which often has an onset in middle childhood and adolescence, is a major risk factor for mental health difficulties. Therefore, treatments targeting underlying and potentially modifiable mechanisms rather than anxiety symptoms may be more likely to be effective.

A microRNA-7/growth arrest specific 6/TYRO3 axis regulates the growth and invasiveness of sorafenib-resistant cells in human hepatocellular carcinoma.

Sorafenib remains the only approved drug for treating patients with advanced hepatocellular carcinoma (HCC). However, the therapeutic effect of sorafenib is transient, and patients invariably develop sorafenib resistance (SR). Recently, TYRO3, a member of the TYRO3-AXL-MER family of receptor tyrosine kinases, was identified as being aberrantly expressed in a significant proportion of HCC; however, its role in SR is unknown. In this study, we generated two functionally distinct sorafenib-resistant human Huh-7 HCC cell lines in order to identify new mechanisms to abrogate acquired SR as well as new potential therapeutic targets in HCC. Initially, we investigated the effects of a microRNA (miR), miR-7-5p (miR-7), in both in vitro and in vivo preclinical models of human HCC and identified miR-7 as a potent tumor suppressor of human HCC. We identified TYRO3 as a new functional target of miR-7, which regulates proliferation, migration, and invasion of Huh-7 cells through the phosphoinositide 3-kinase/protein kinase B pathway and is markedly elevated with acquisition of SR. Furthermore, miR-7 effectively silenced TYRO3 expression in both sorafenib-sensitive and sorafenib-resistant Huh-7 cells, inhibiting TYRO3/growth arrest specific 6-mediated cancer cell migration and invasion. CONCLUSION: We identified a mechanism for acquiring SR in HCC that is through the aberrant expression of the TYRO3/phosphoinositide 3-kinase/protein kinase B signal transduction pathway, and that can be overcome by miR-7 overexpression. Taken together, these data suggest a potential role for miR-7 as an RNA-based therapeutic to treat refractory and drug-resistant HCC. (Hepatology 2018;67:216-231).

RNA-induced silencing complex (RISC) Proteins PACT, TRBP, and Dicer are SRA binding nuclear receptor coregulators.

The cytoplasmic RNA-induced silencing complex (RISC) contains dsRNA binding proteins, including protein kinase RNA activator (PACT), transactivation response RNA binding protein (TRBP), and Dicer, that process pre-microRNAs into mature microRNAs (miRNAs) that target specific mRNA species for regulation. There is increasing evidence for important functional interactions between the miRNA and nuclear receptor (NR) signaling networks, with recent data showing that estrogen, acting through the estrogen receptor, can modulate initial aspects of nuclear miRNA processing. Here, we show that the cytoplasmic RISC proteins PACT, TRBP, and Dicer are steroid receptor RNA activator (SRA) binding NR coregulators that target steroid-responsive promoters and regulate NR activity and downstream gene expression. Furthermore, each of the RISC proteins, together with Argonaute 2, associates with SRA and specific pre-microRNAs in both the nucleus and cytoplasm, providing evidence for links between NR-mediated transcription and some of the factors involved in miRNA processing.

A MYC-ZNF148-ID1/3 regulatory axis modulating cancer stem cell traits in aggressive breast cancer.

The MYC proto-oncogene (MYC) is one of the most frequently overexpressed genes in breast cancer that drives cancer stem cell-like traits, resulting in aggressive disease progression and poor prognosis. In this study, we identified zinc finger transcription factor 148 (ZNF148, also called Zfp148 and ZBP-89) as a direct target of MYC. ZNF148 suppressed cell proliferation and migration and was transcriptionally repressed by MYC in breast cancer. Depletion of ZNF148 by short hairpin RNA (shRNA) and CRISPR/Cas9 increased triple-negative breast cancer (TNBC) cell proliferation and migration. Global transcriptome and chromatin occupancy analyses of ZNF148 revealed a central role in inhibiting cancer cell de-differentiation and migration. Mechanistically, we identified the Inhibitor of DNA binding 1 and 3 (ID1, ID3), drivers of cancer stemness and plasticity, as previously uncharacterized targets of transcriptional repression by ZNF148. Silencing of ZNF148 increased the stemness and tumorigenicity in TNBC cells. These findings uncover a previously unknown tumor suppressor role for ZNF148, and a transcriptional regulatory circuitry encompassing MYC, ZNF148, and ID1/3 in driving cancer stem cell traits in aggressive breast cancer.

The tumor suppressor miR-642a-5p targets Wilms Tumor 1 gene and cell-cycle progression in prostate cancer.

RNA-based therapeutics are emerging as innovative options for cancer treatment, with microRNAs being attractive targets for therapy development. We previously implicated microRNA-642a-5p (miR-642a-5p) as a tumor suppressor in prostate cancer (PCa), and here we characterize its mode of action, using 22Rv1 PCa cells. In an in vivo xenograft tumor model, miR-642a-5p induced a significant decrease in tumor growth, compared to negative control. Using RNA-Sequencing, we identified gene targets of miR-642a-5p which were enriched for gene sets controlling cell cycle; downregulated genes included Wilms Tumor 1 gene (WT1), NUAK1, RASSF3 and SKP2; and upregulated genes included IGFBP3 and GPS2. Analysis of PCa patient datasets showed a higher expression of WT1, NUAK1, RASSF3 and SKP2; and a lower expression of GPS2 and IGFBP3 in PCa tissue compared to non-malignant prostate tissue. We confirmed the prostatic oncogene WT1, as a direct target of miR-642a-5p, and treatment of 22Rv1 and LNCaP PCa cells with WT1 siRNA or a small molecule inhibitor of WT1 reduced cell proliferation. Taken together, these data provide insight into the molecular mechanisms by which miR-642a-5p acts as a tumor suppressor in PCa, an effect partially mediated by regulating genes involved in cell cycle control; and restoration of miR-642-5p in PCa could represent a novel therapeutic approach.

Label-free quantitative analysis of one-dimensional PAGE LC/MS/MS proteome: application on angiotensin II-stimulated smooth muscle cells secretome.

A widely used method for protein identification couples prefractionation of protein samples by one-dimensional (1D) PAGE with LC/MS/MS. We developed a new label-free quantitative algorithm by combining measurements of spectral counting, ion intensity, and peak area on 1D PAGE-based proteomics. This algorithm has several improvements over other label-free quantitative algorithms: (i) Errors in peak detection are reduced because the retention time is based on each LC/MS/MS run and actual precursor m/z. (ii) Detection sensitivity is increased because protein quantification is based on the combination of peptide count, ion intensity, and peak area. (iii) Peak intensity and peak area are calculated in each LC/MS/MS run for all slices from 1D PAGE for every single identified protein and visualized as a Western blot image. The sensitivity and accuracy of this algorithm were demonstrated by using standard curves (17.4 fmol to 8.7 pmol), complex protein mixtures (30 fmol to 1.16 pmol) of known composition, and spiked protein (34.8 fmol to 17.4 pmol) in complex proteins. We studied the feasibility of this approach using the secretome of angiotensin II (Ang II)-stimulated vascular smooth muscle cells (VSMCs). From the VSMC-conditioned medium, 629 proteins were identified including 212 putative secreted proteins. 26 proteins were differently expressed in control and Ang II-stimulated VSMCs, including 18 proteins not previously reported. Proteins related to cell growth (CYR61, protein NOV, and clusterin) were increased, whereas growth arrest-specific 6 (GAS6) and growth/differentiation factor 6 were decreased by Ang II stimulation. Ang II-stimulated changes of plasminogen activator inhibitor-1, GAS6, cathepsin B, and periostin were validated by Western blot. In conclusion, a novel label-free quantitative analysis of 1D PAGE-LC/MS/MS-based proteomics has been successfully applied to the identification of new potential mediators of Ang II action and may provide an alternative to traditional protein staining methods.

Evaluation of MicroRNA Delivery In Vivo.

MicroRNAs (miRNAs) are a family of short noncoding RNA molecules that fine-tune expression of mRNAs. Often their altered expression is associated with a number of diseases, including cancer. Given that miRNAs target multiple genes and "difficult to drug" oncogenes, they present attractive candidates to manipulate as an anti-cancer strategy. MicroRNA-7 (miR-7) is a tumor suppressor miRNA that has been shown to target oncogenes overexpressed in cancers, such as the epidermal growth factor receptor (EGFR) and the nuclear factor-κ B subunit, RelA. Here, we describe methods for evaluating systemic delivery of miR-7 using a lipid nanoparticle formulation in an animal model. The microRNA is delivered three times, over 1 week and tissues collected 24 h after the last injection. RNA and protein are extracted from snap frozen tissues and processed to detect miRNA distribution and subsequent assessment of downstream targets and signaling mediators, respectively. Importantly, variability in efficiency of miRNA delivery will be observed between organs of the same animal and also between animals. Additionally, delivering the microRNA to organs other than the liver, particularly the brain, remains challenging. Furthermore, large variation in miRNA targets is seen both within tissues and across tissues depending on the lysis buffer used for protein extraction. Therefore, analyzing protein expression is dependent upon the method used for isolation and requires optimization for each individual application. Together, these methods will provide a foundation for those planning on assessing the efficacy of delivery of a miRNA in vivo.

miR-331-3p and Aurora Kinase inhibitor II co-treatment suppresses prostate cancer tumorigenesis and progression.

RNA-based therapeutics could represent a new avenue of cancer treatment. miRNA 331-3p (miR-331-3p) is implicated in prostate cancer (PCa) as a putative tumor suppressor, but its functional activity and synergy with other anti-tumor agents is largely unknown. We found miR-331-3p expression in PCa tumors was significantly decreased compared to non-malignant matched tissue. Analysis of publicly available PCa gene expression data sets showed miR-331-3p expression negatively correlated with Gleason Score, tumor stage, lymph node involvement and PSA value, and was significantly down regulated in tumor tissue relative to normal prostate tissue. Overexpression of miR-331-3p reduced PCa cell growth, migration and colony formation, as well as xenograft tumor initiation, proliferation and survival of mice. Microarray analysis identified seven novel targets of miR-331-3p in PCa. The 3'-untranslated regions of PLCγ1 and RALA were confirmed as targets of miR-331-3p, with mutation analyses confirming RALA as a direct target. Expression of miR-331-3p or RALA siRNA in PCa cells reduced RALA expression, proliferation, migration and colony formation in vitro. RALA expression positively correlated with Gleason grade in two separate studies, as well as in a PCa tissue microarray. Co-treatment using siRALA with an Aurora Kinase inhibitor (AKi-II) decreased colony formation of PCa cells while the combination of AKi-II with miR-331-3p resulted in significant reduction of PCa cell proliferation in vitro and PCa xenograft growth in vivo. Thus, miR-331-3p directly targets the RALA pathway and the addition of the AKi-II has a synergistic effect on tumor growth inhibition, suggesting a potential role as combination therapy in PCa.

Overexpression and knock-down studies highlight that a disintegrin and metalloproteinase 28 controls proliferation and migration in human prostate cancer.

Prostate cancer is one of the most prevalent cancers in men. It is critical to identify and characterize oncogenes that drive the pathogenesis of human prostate cancer. The current study builds upon previous research showing that a disintegrin and metallproteinase (ADAM)28 is involved in the pathogenesis of numerous cancers. Our novel study used overexpression, pharmacological, and molecular approaches to investigate the biological function of ADAM28 in human prostate cancer cells, with a focus on cell proliferation and migration. The results of this study provide important insights into the role of metalloproteinases in human prostate cancer.The expression of ADAM28 protein levels was assessed within human prostate tumors and normal adjacent tissue by immunohistochemistry. Immunocytochemistry and western blotting were used to assess ADAM28 protein expression in human prostate cancer cell lines. Functional assays were conducted to assess proliferation and migration in human prostate cancer cells in which ADAM28 protein expression or activity had been altered by overexpression, pharmacological inhibition, or by siRNA gene knockdown.The membrane bound ADAM28 was increased in human tumor biopsies and prostate cancer cell lines. Pharmacological inhibition of ADAM28 activity and/or knockdown of ADAM28 significantly reduced proliferation and migration of human prostate cancer cells, while overexpression of ADAM28 significantly increased proliferation and migration.ADAM28 is overexpressed in primary human prostate tumor biopsies, and it promotes human prostate cancer cell proliferation and migration. This study supports the notion that inhibition of ADAM28 may be a potential novel therapeutic strategy for human prostate cancer.

microRNA-7-5p inhibits melanoma cell proliferation and metastasis by suppressing RelA/NF-κB.

microRNA-7-5p (miR-7-5p) is a tumor suppressor in multiple cancer types and inhibits growth and invasion by suppressing expression and activity of the epidermal growth factor receptor (EGFR) signaling pathway. While melanoma is not typically EGFR-driven, expression of miR-7-5p is reduced in metastatic tumors compared to primary melanoma. Here, we investigated the biological and clinical significance of miR-7-5p in melanoma. We found that augmenting miR-7-5p expression in vitro markedly reduced tumor cell viability, colony formation and induced cell cycle arrest. Furthermore, ectopic expression of miR-7-5p reduced migration and invasion of melanoma cells in vitro and reduced metastasis in vivo. We used cDNA microarray analysis to identify a subset of putative miR-7-5p target genes associated with melanoma and metastasis. Of these, we confirmed nuclear factor kappa B (NF-κB) subunit RelA, as a novel direct target of miR-7-5p in melanoma cells, such that miR-7-5p suppresses NF-κB activity to decrease expression of canonical NF-κB target genes, including IL-1ß, IL-6 and IL-8. Importantly, the effects of miR-7-5p on melanoma cell growth, cell cycle, migration and invasion were recapitulated by RelA knockdown. Finally, analysis of gene array datasets from multiple melanoma patient cohorts revealed an association between elevated RelA expression and poor survival, further emphasizing the clinical significance of RelA and its downstream signaling effectors. Taken together, our data show that miR-7-5p is a potent inhibitor of melanoma growth and metastasis, in part through its inactivation of RelA/NF-κB signaling. Furthermore, miR-7-5p replacement therapy could have a role in the treatment of this disease.

Axl mediates acquired resistance of head and neck cancer cells to the epidermal growth factor receptor inhibitor erlotinib.

Elevated expression and activity of the epidermal growth factor receptor (EGFR) is associated with development and progression of head and neck cancer (HNC) and a poor prognosis. Clinical trials with EGFR tyrosine kinase inhibitors (e.g., erlotinib) have been disappointing in HNC. To investigate the mechanisms mediating resistance to these agents, we developed an HNC cell line (HN5-ER) with acquired erlotinib resistance. In contrast to parental HN5 HNC cells, HN5-ER cells exhibited an epithelial-mesenchymal (EMT) phenotype with increased migratory potential, reduced E-cadherin and epithelial-associated microRNAs (miRNA), and elevated vimentin expression. Phosphorylated receptor tyrosine kinase profiling identified Axl activation in HN5-ER cells. Growth and migration of HN5-ER cells were blocked with a specific Axl inhibitor, R428, and R428 resensitized HN5-ER cells to erlotinib. Microarray analysis of HN5-ER cells confirmed the EMT phenotype associated with acquired erlotinib resistance, and identified activation of gene expression associated with cell migration and inflammation pathways. Moreover, increased expression and secretion of interleukin (IL)-6 and IL-8 in HN5-ER cells suggested a role for inflammatory cytokine signaling in EMT and erlotinib resistance. Expression of the tumor suppressor miR-34a was reduced in HN5-ER cells and increasing its expression abrogated Axl expression and reversed erlotinib resistance. Finally, analysis of 302 HNC patients revealed that high tumor Axl mRNA expression was associated with poorer survival (HR = 1.66, P = 0.007). In summary, our results identify Axl as a key mediator of acquired erlotinib resistance in HNC and suggest that therapeutic inhibition of Axl by small molecule drugs or specific miRNAs might overcome anti-EGFR therapy resistance.

SLIRP, a small SRA binding protein, is a nuclear receptor corepressor.

Steroid receptor RNA activator (SRA), the only known RNA coactivator, augments transactivation by nuclear receptors (NRs). We identified SLIRP (SRA stem-loop interacting RNA binding protein) binding to a functional substructure of SRA, STR7. SLIRP is expressed in normal and tumor tissues, contains an RNA recognition motif (RRM), represses NR transactivation in a SRA- and RRM-dependent manner, augments the effect of Tamoxifen, and modulates association of SRC-1 with SRA. SHARP, a RRM-containing corepressor, also binds STR7, augmenting repression with SLIRP. SLIRP colocalizes with SKIP (Chr14q24.3), another NR coregulator, and reduces SKIP-potentiated NR signaling. SLIRP is recruited to endogenous promoters (pS2 and metallothionein), the latter in a SRA-dependent manner, while NCoR promoter recruitment is dependent on SLIRP. The majority of the endogenous SLIRP resides in the mitochondria. Our data demonstrate that SLIRP modulates NR transactivation, suggest it may regulate mitochondrial function, and provide mechanistic insight into interactions between SRA, SLIRP, SRC-1, and NCoR.