Transcription factor network analysis based on single cell RNA-seq identifies that Trichostatin-a reverses docetaxel resistance in prostate Cancer.

BACKGROUND: Overcoming drug resistance is critical for increasing the survival rate of prostate cancer (PCa). Docetaxel is the first cytotoxic chemotherapeutical approved for treatment of PCa. However, 99% of PCa patients will develop resistance to docetaxel within 3 years. Understanding how resistance arises is important to increasing PCa survival. METHODS: In this study, we modeled docetaxel resistance using two PCa cell lines: DU145 and PC3. Using the Passing Attributes between Networks for Data Assimilation (PANDA) method to model transcription factor (TF) activity networks in both sensitive and resistant variants of the two cell lines. We identified edges and nodes shared by both PCa cell lines that composed a shared TF network that modeled changes which occur during acquisition of docetaxel resistance in PCa. We subjected the shared TF network to connectivity map analysis (CMAP) to identify potential drugs that could disrupt the resistant networks. We validated the candidate drug in combination with docetaxel to treat docetaxel-resistant PCa in both in vitro and in vivo models. RESULTS: In the final shared TF network, 10 TF nodes were identified as the main nodes for the development of docetaxel resistance. CMAP analysis of the shared TF network identified trichostatin A (TSA) as a candidate adjuvant to reverse docetaxel resistance. In cell lines, the addition of TSA to docetaxel enhanced cytotoxicity of docetaxel resistant PCa cells with an associated reduction of the IC50 of docetaxel on the resistant cells. In the PCa mouse model, combination of TSA and docetaxel reduced tumor growth and final weight greater than either drug alone or vehicle. CONCLUSIONS: We identified a shared TF activity network that drives docetaxel resistance in PCa. We also demonstrated a novel combination therapy to overcome this resistance. This study highlights the usage of novel application of single cell RNA-sequencing and subsequent network analyses that can reveal novel insights which have the potential to improve clinical outcomes.

Utilizing online tools to increase volunteer ombudsmen presence in long-term care.

In this descriptive study, former and current volunteer ombudsmen (n = 65) completed an online survey and Chi-square analyses were used to determine group differences in order to examine the impact of internet-based communication on the recruitment and retention of volunteer long-term care ombudsmen. The results showed that the program's shift to internet-based recruitment and communication methods helped increase the number of volunteers by 50% and contributed to a positive shift in role perception and satisfaction. Consequently, the proliferation of internet and social media usage permits greater volunteer management opportunities than previously were available. These tools also allow for consistency of message, extended training opportunities, and recourse to resources at need which permit ombudsmen volunteers to identify more readily with the role of resident advocate and receive greater performance satisfaction as it relates to that role.

Urinary extracellular vesicle-derived miR-126-3p predicts lymph node invasion in patients with high-risk prostate cancer.

To investigate extracellular vesicles (EVs), biomarkers for predicting lymph node invasion (LNI) in patients with high-risk prostate cancer (HRPCa), plasma, and/or urine samples were prospectively collected from 45 patients with prostate cancer (PCa) and five with benign prostatic hyperplasia (BPH). Small RNA sequencing was performed to identify miRNAs in the EVs. All patients with PCa underwent radical prostatectomy and extended pelvic lymph node dissection. Differentially expressed miRNAs were identified in patients with and without pathologically-verified LNI. The candidate miRNAs were validated in low-risk prostate cancer (LRPCa) and BPH. Four miRNA species (e.g., miR-126-3p) and three miRNA species (e.g., miR-27a-3p) were more abundant in urinary and plasma EVs, respectively, of patients with PCa. None of these miRNA species were shared between urinary and plasma EVs. miR-126-3p was significantly more abundant in patients with HR PCa with LNI than in those without (P = 0.018). miR-126-3p was significantly more abundant in the urinary EVs of patients with HRPCa than in those with LRPCa (P = 0.017) and BPH (P = 0.011). In conclusion, urinary EVs-derived miR-126-3p may serve as a good biomarker for predicting LNI in patients with HRPCa.

Urinary extracellular vesicle-derived miR-126-3p predicts lymph node invasion in patients with high-risk prostate cancer.

To investigate extracellular vesicles (EVs) biomarkers for predicting lymph node invasion (LNI) in patients with high-risk prostate cancer (HRPCa), plasma and/or urine samples were prospectively collected from 45 patients with prostate cancer (PCa) and five with benign prostatic hyperplasia (BPH). Small RNA sequencing was performed to identify miRNAs in the EVs. All patients with PCa underwent radical prostatectomy and extended pelvic lymph node dissection. Differentially-expressed miRNAs were identified in patients with and without pathologically-verified LNI. The candidate miRNAs were validated in low-risk prostate cancer (LRPCa) and BPH. Four miRNA species (e.g. miR-126-3p) and three miRNA species (e.g. miR-27a-3p) were more abundant in urinary and plasma EVs, respectively, of patients with PCa. None of these miRNA species were shared between urinary and plasma EVs. miR-126-3p was significantly more abundant in patients with HR PCa with LNI than in those without (P = 0.018). miR-126-3p was significantly more abundant in the urinary EVs of patients with HRPCa than in those with LRPCa (P = 0.017) and BPH (P = 0.011). In conclusion, urinary EVs-derived miR-126-3p may serve as a good biomarker for predicting LNI in patients with HRPCa.

Integration of Single-Cell RNA-Sequencing and Network Analysis to Investigate Mechanisms of Drug Resistance.

Innate resistance and therapeutic-driven development of resistance to anticancer drugs is a common complication of cancer therapy. Understanding mechanisms of drug resistance can lead to development of alternative therapies. One strategy is to subject drug-sensitive and drug-resistant variants to single-cell RNA-seq (scRNA-seq) and to subject the scRNA-seq data to network analysis to identify pathways associated with drug resistance. This protocol describes a computational analysis pipeline to study drug resistance by subjecting scRNA-seq expression data to Passing Attributes between Networks for Data Assimilation (PANDA), an integrative network analysis tool that incorporates protein-protein interactions (PPI) and transcription factor (TF)-binding motifs.

Pheno-SELEX: Engineering Anti-Metastatic Aptamers through Targeting the Invasive Phenotype Using Systemic Evolution of Ligands by Exponential Enrichment.

Multiple methods (e.g., small molecules and antibodies) have been engineered to target specific proteins and signaling pathways in cancer. However, many mediators of the cancer phenotype are unknown and the ability to target these phenotypes would help mitigate cancer. Aptamers are small DNA or RNA molecules that are designed for therapeutic use. The design of aptamers to target cancers can be challenging. Accordingly, to engineer functionally anti-metastatic aptamers we used a modification of systemic evolution of ligands by exponential enrichment (SELEX) we call Pheno-SELEX to target a known phenotype of cancer metastasis, i.e., invasion. A highly invasive prostate cancer (PCa) cell line was established and used to identify aptamers that bound to it with high affinity as opposed to a less invasive variant to the cell line. The anti-invasive aptamer (AIA1) was found to inhibit in vitro invasion of the original highly invasive PCa cell line, as well as an additional PCa cell line and an osteosarcoma cell line. AIA1 also inhibited in vivo development of metastasis in both a PCa and osteosarcoma model of metastasis. These results indicate that Pheno-SELEX can be successfully used to identify aptamers without knowledge of underlying molecular targets. This study establishes a new paradigm for the identification of functional aptamers.

IgV somatic mutation of human anti-SARS-CoV-2 monoclonal antibodies governs neutralization and breadth of reactivity.

Abs that neutralize SARS-CoV-2 are thought to provide the most immediate and effective treatment for those severely afflicted by this virus. Because coronavirus potentially diversifies by mutation, broadly neutralizing Abs are especially sought. Here, we report a possibly novel approach to rapid generation of potent broadly neutralizing human anti-SARS-CoV-2 Abs. We isolated SARS-CoV-2 spike protein-specific memory B cells by panning from the blood of convalescent subjects after infection with SARS-CoV-2 and sequenced and expressed Ig genes from individual B cells as human mAbs. All of 43 human mAbs generated in this way neutralized SARS-CoV-2. Eighteen of the forty-three human mAbs exhibited half-maximal inhibitory concentrations (IC50) of 6.7 × 10-12 M to 6.7 × 10-15 M for spike-pseudotyped virus. Seven of the human mAbs also neutralized (with IC50 < 6.7 × 10-12 M) viruses pseudotyped with mutant spike proteins (including receptor-binding domain mutants and the S1 C-terminal D614G mutant). Neutralization of the Wuhan Hu-1 founder strain and of some variants decreased when coding sequences were reverted to germline, suggesting that potency of neutralization was acquired by somatic hypermutation and selection of B cells. These results indicate that infection with SARS-CoV-2 evokes high-affinity B cell responses, some products of which are broadly neutralizing and others highly strain specific. We also identify variants that would potentially resist immunity evoked by infection with the Wuhan Hu-1 founder strain or by vaccines developed with products of that strain, suggesting evolutionary courses that SARS-CoV-2 could take.

Single-Cell Transcriptomics Analysis Identifies Nuclear Protein 1 as a Regulator of Docetaxel Resistance in Prostate Cancer Cells.

The majority of patients with prostate cancer treated with docetaxel develop resistance to it. To better understand the mechanism behind the acquisition of resistance, we conducted single-cell RNA-sequencing (scRNA-seq) of docetaxel-sensitive and -resistant variants of DU145 and PC3 prostate cancer cell lines. Overall, sensitive and resistant cells clustered separately. Differential gene expression analysis between resistant and sensitive cells revealed 182 differentially expressed genes common to both prostate cancer cell lines. A subset of these genes gave a gene expression profile in the resistant transcriptome-like-sensitive cells similar to the resistant cells. Exploration for functional gene pathways identified 218 common pathways between the two cell lines. Protein ubiquitination was the most differentially regulated pathway and was enriched in the resistant cells. Transcriptional regulator analysis identified 321 potential regulators across both cell lines. One of the top regulators identified was nuclear protein 1 (NUPR1). In contrast to the single-cell analysis, bulk analysis of the cells did not reveal NUPR1 as a promising candidate. Knockdown and overexpression of NUPR1 in the prostate cancer cells demonstrated that NUPR1 confers docetaxel resistance in both cell lines. Collectively, these data demonstrate the utility of scRNA-seq to identify regulators of drug resistance. Furthermore, NUPR1 was identified as a mediator of prostate cancer drug resistance, which provides the rationale to explore NUPR1 and its target genes for reversal of docetaxel resistance. IMPLICATIONS: Using single-cell sequencing of prostate cancer, we show that NUPR1 plays a role in docetaxel resistance.

Efficacy and Effect of Cabozantinib on Bone Metastases in Treatment-naive Castration-resistant Prostate Cancer.

BACKGROUND: Cabozantinib is active in advanced prostate cancer with improvement on bone scans in men on phase II trials. This trial evaluated the efficacy and changes in bone lesions in men with metastatic castration-resistant prostate cancer (mCRPC) treated with cabozantinib. PATIENTS AND METHODS: Eligible patients with mCRPC involving bone underwent biopsy of a bone lesion followed by cabozantinib starting at 60 mg daily and continuing until progression or intolerable toxicity. The primary study endpoint was progression-free survival at 12 weeks. The bone lesion was rebiopsied at 6 weeks. Expression of CMET, phospho-CMET, and VEGFR2 was assayed by immunohistochemistry. Serum was obtained at baseline, and at 3, 6, and 12 weeks and assayed for bone remodeling markers. RESULTS: A total of 25 patients were enrolled: 22 were evaluable, and 3 were excluded before receiving cabozantinib. At 12 weeks, 17 (77%) of 22 patients had stable disease or better. The median time on treatment was 24 weeks (range, 3-112 weeks). The overall median progression-free survival was 43.7 weeks (95% confidence interval, 23.7-97.0 weeks). Eight (36%) of 22 patients had markedly reduced uptake on bone scan. Patients with significant response on bone scan had higher bone morphogenic protein-2 levels at baseline, stable N-telopeptides levels, increased vascular endothelial growth factor receptor 2 expression, and a trend towards increased phospho-CMET while on cabozantinib compared with patients with stable disease. CONCLUSIONS: Cabozantinib is active in men with mCRPC, inducing significant changes on bone scan in one-third of patients with changes in markers of bone formation and the tumor microenvironment.

Abituzumab Targeting of αV-Class Integrins Inhibits Prostate Cancer Progression.

Integrins that contain an integrin αV subunit contribute to multiple functions that promote cancer progression. The goal of this study was to determine whether abituzumab (DI17E6, EMD 525797), a humanized monoclonal antibody (mAb) against integrin αV impacts, prostate cancer progression. To evaluate this, prostate cancer cells were treated with DI17E6 and its effects on proliferation, apoptosis, cell-cycle, adhesion, detachment, migration, invasion and phosphorylation of downstream targets, including FAK, Akt, and ERK, were determined. DI17E6 promoted detachment and inhibited adhesion of prostate cancer cells to several extracellular matrix (ECM) proteins and cells found in the bone microenvironment but had no impact on cell viability, cell-cycle, and caspase-3/7 activity. DI17E6 inhibited migration and invasion of prostate cancer cells. In addition, DI7E6 decreased phosphorylation of FAK, Akt, and ERK. These results indicate that inhibition of integrin αV with DI17E6 inhibits several prometastatic phenotypes of prostate cancer cells and therefore provide a rationale for further evaluation of DI17E6 for diminishing prostate cancer progression.Implications: This work identifies that therapeutic targeting of integrins containing an αV integrin unit inhibits cancer progression and thus may be of clinical benefit. Mol Cancer Res; 15(7); 875-83. ©2017 AACR.

Psychophysics in functional strength and power training: review and implementation framework.

Functional training is considered to be training that attempts to mimic the specific physiological demands of real-life activities. Most approaches to functional training, though, omit important factors that contribute to physiological and neuromotor adaptations. Cognitive factors related to sports influence physiological performance, and subsequently, physiological and neuromotor adaptations. We present a rationale and a theoretical framework by which to create effective functional training methods that incorporate cognitive factors. This framework draws upon recent developments and strong empirical evidence in the areas of dynamic systems theory, perceptual skills training, and motor learning/control. Emphasized within rigorous physical training are practice-related techniques and motor-learning strategies. In particular, mental effort, attention, and intention manipulated in a discovery-learning paradigm provide a framework for functional strength and power training. This framework is suggested to help maximize sport-specific physiological adaptations, and subsequently, sports performance.

Magnaporthe grisea interactions with the model grass Brachypodium distachyon closely resemble those with rice (Oryza sativa).

SUMMARY Germplasm of Brachypodium distachyon was inoculated with Magnaporthe grisea using either rice- (Guy11) or grass-adapted (FAG1.1.1, PA19w-06, PA31v-01) host-limited forms of the fungus, and interactions with varying degrees of susceptibility and resistance were identified. Ecotype ABR5 was resistant to each M. grisea strain whereas ABR1 was susceptible to all but P31vi-01. Mendelian segregation in ABR1 x ABR5 crosses suggested that a single dominant resistance gene conferred resistance to Guy11. Microscopic analyses revealed that the aetiology of Guy11 fungal development and disease progression in ABR1 closely resembled that of rice infections. In ABR5, Guy11 pathogenesis was first suppressed at 48 h post-inoculation, at the secondary hyphal formation stage and was coincident with cytoplasmic granulation. Resistance to strains PA31v-01 and FAG1.1.1 was associated with a localized cell death with little callose deposition. 3,3-Diaminobenzidine staining indicated the elicitation of cell death in B. distachyon was preceded by oxidative stress in the interacting epidermal cells and the underlying mesophyll cells. Northern blot hybridization using probes for barley genes (PR1, PR5 and PAL) indicated that each was more rapidly expressed in ABR5 challenged with Guy11 although the B. distachyon defence genes BD1 and BD8 were more quickly induced in ABR1. Such data show that B. distachyon is an appropriate host for functional genomic investigations into M. grisea pathology and plant responses.