Insights from HuR biology point to potential improvement for second-line ovarian cancer therapy.

This retrospective study aimed to investigate the role that an RNA-binding protein, HuR, plays in the response of high-grade serous ovarian tumors to chemotherapeutics. We immunohistochemically stained sections of 31 surgically-debulked chemo-naïve ovarian tumors for HuR and scored the degree of HuR cytoplasmic staining. We found no correlation between HuR intracellular localization in tumor sections and progression free survival (PFS) of these patients, 29 of whom underwent second-line gemcitabine/platin combination therapy for recurrent disease. Ribonucleoprotein immunoprecipitation (RNP-IP) analysis of ovarian cancer cells in culture showed that cytoplasmic HuR increases deoxycytidine kinase (dCK), a metabolic enzyme that activates gemcitabine. The effects of carboplatin treatment on HuR and WEE1 (a mitotic inhibitor) expression, and on cell cycle kinetics, were also examined. Treatment of ovarian cancer cells with carboplatin results in increased HuR cytoplasmic expression and elevated WEE1 expression, arresting cell cycle G2/M transition. This may explain why HuR cytoplasmic localization in chemo-naïve tumors is not predictive of therapeutic response and PFS following second-line gemcitabine/platin combination therapy. These results suggest treatment of recurrent ovarian tumors with a combination of gemcitabine, carboplatin, and a WEE1 inhibitor may be potentially advantageous as compared to current clinical practices

Posttranscriptional Upregulation of IDH1 by HuR Establishes a Powerful Survival Phenotype in Pancreatic Cancer Cells.

Cancer aggressiveness may result from the selective pressure of a harsh nutrient-deprived microenvironment. Here we illustrate how such conditions promote chemotherapy resistance in pancreatic ductal adenocarcinoma (PDAC). Glucose or glutamine withdrawal resulted in a 5- to 10-fold protective effect with chemotherapy treatment. PDAC xenografts were less sensitive to gemcitabine in hypoglycemic mice compared with hyperglycemic mice. Consistent with this observation, patients receiving adjuvant gemcitabine (n = 107) with elevated serum glucose levels (HgbA1C \u3e 6.5%) exhibited improved survival. We identified enhanced antioxidant defense as a driver of chemoresistance in this setting. ROS levels were doubled in vitro by either nutrient withdrawal or gemcitabine treatment, but depriving PDAC cells of nutrients before gemcitabine treatment attenuated this effect. Mechanistic investigations based on RNAi or CRISPR approaches implicated the RNA binding protein HuR in preserving survival under nutrient withdrawal, with or without gemcitabine. Notably, RNA deep sequencing and functional analyses in HuR-deficient PDAC cell lines identified isocitrate dehydrogenase 1 (IDH1) as the sole antioxidant enzyme under HuR regulation. HuR-deficient PDAC cells lacked the ability to engraft successfully in immunocompromised mice, but IDH1 overexpression in these cells was sufficient to fully restore chemoresistance under low nutrient conditions. Overall, our findings highlight the HuR–IDH1 regulatory axis as a critical, actionable therapeutic target in pancreatic cancer

Targeting the mRNA-binding protein HuR impairs malignant characteristics of pancreatic ductal adenocarcinoma cells.

Post-transcriptional regulation is a powerful mediator of gene expression, and can rapidly alter the expression of numerous transcripts involved in tumorigenesis. We have previously shown that the mRNA-binding protein HuR (ELAVL1) is elevated in human pancreatic ductal adenocarcinoma (PDA) specimens compared to normal pancreatic tissues, and its cytoplasmic localization is associated with increased tumor stage. To gain a better insight into HuR\u27s role in PDA biology and to assess it as a candidate therapeutic target, we altered HuR expression in PDA cell lines and characterized the resulting phenotype in preclinical models. HuR silencing by short hairpin and small interfering RNAs significantly decreased cell proliferation and anchorage-independent growth, as well as impaired migration and invasion. In comparison, HuR overexpression increased migration and invasion, but had no significant effects on cell proliferation and anchorage-independent growth. Importantly, two distinct targeted approaches to HuR silencing showed marked impairment in tumor growth in mouse xenografts. NanoString nCounter® analyses demonstrated that HuR regulates core biological processes, highlighting that HuR inhibition likely thwarts PDA viability through post-transcriptional regulation of diverse signaling pathways (e.g. cell cycle, apoptosis, DNA repair). Taken together, our study suggests that targeted inhibition of HuR may be a novel, promising approach to the treatment of PDA

Analysis of 13 cell types reveals evidence for the expression of numerous novel primate- and tissue-specific microRNAs

MicroRNAs (miRNAs) are small ∼22-nt RNAs that are important regulators of posttranscriptional gene expression. Since their initial discovery, they have been shown to be involved in many cellular processes, and their misexpression is associated with disease etiology. Currently, nearly 2,800 human miRNAs are annotated in public repositories. A key question in miRNA research is how many miRNAs are harbored by the human genome. To answer this question, we examined 1,323 short RNA sequence samples and identified 3,707 novel miRNAs, many of which are human-specific and tissue-specific. Our findings suggest that the human genome expresses a greater number of miRNAs than has previously been appreciated and that many more miRNA molecules may play key roles in disease etiology

Targeting the mRNA-binding protein HuR impairs malignant characteristics of pancreatic ductal adenocarcinoma cells

Post-transcriptional regulation is a powerful mediator of gene expression, and can rapidly alter the expression of numerous transcripts involved in tumorigenesis. We have previously shown that the mRNA-binding protein HuR (ELAVL1) is upregulated in human pancreatic ductal adenocarcinoma (PDA) specimens compared to normal pancreatic tissues, and its cytoplasmic localization is associated with increased tumor stage. To gain a better insight into HuR’s role in PDA biology and to assess it as a candidate therapeutic target, we altered HuR expression in PDA cell lines and characterized the resulting phenotype in preclinical models. HuR silencing by short hairpin and small interfering RNAs significantly decreased cell proliferation and anchorage-independent growth, as well as impaired migration and invasion. In comparison, HuR overexpression increased migration and invasion, but had no significant effects on cell proliferation and anchorage-independent growth. Importantly, two distinct targeted approaches to HuR silencing showed marked impairment in tumor growth in mouse xenografts. NanoString nCounter® analyses demonstrated that HuR regulates core biological processes, highlighting that HuR inhibition likely thwarts PDA viability through post-transcriptional regulation of diverse signaling pathways (e.g. cell cycle, apoptosis, DNA repair). Taken together, our study suggests that targeting HuR may be a novel, promising approach to the treatment of PDA

Penile prosthesis implantation technique for patients with a neophallus

Introduction and objective: Erectile function is an important goal for many patients post-phalloplasty. Our objective is to demonstrate a technique for placement of a single-cylinder inflatable penile prosthesis (IPP) in patients with a neophallus. Surgical procedure: IPP placement is performed 1-2 years post-phalloplasty for optimal return of sensation and vascularization. The patient voids preoperatively. Since the bladder is emptied immediately before surgery, we opt not to place a catheter in the potentially tortuous urethra and place the reservoir as early as possible to avoid significant bladder filling, which decreases the risk of bladder injury. Cefazolin and Gentamicin are administered preoperatively and a low concentration Chlorhexidine Gluconate solution is used for irrigation. An infrapubic incision is made and dissection is carried down to the pubic symphysis. The reservoir is placed in the midline prevesical space. Sharp dissection is used to develop a space in the dorsal midline of the neophallus opposite the neourethra, which is palpable without a catheter, prior to sequential distal dilation. After measuring proximally and distally, the appropriately sized device is selected and modified with a shortened, blunted tip and proximal synthetic graft. Previously placed 3–0 FiberWire anchoring sutures on the pubic bone are secured to the proximal aspect of the device. The device is then inflated to facilitate placement into the dilated space distally before tying down the sutures. Pump placement proceeds contralateral to the vascular pedicle in the most dependent aspect of the neoscrotum. Once the tubing is connected, device cycling is performed to ensure proper placement and functioning. Results: Patients are discharged the same day with the device 60% inflated. Sexual activity may occur as early as 8 weeks postoperatively. Complications are seen in up to 40% of patients. Conclusion: Continued improvements in devices and techniques are necessary to decrease complications and improve surgical outcomes as more patients pursue IPP placement post-phalloplasty

Gemcitabine-loaded microbubble system for ultrasound imaging and therapy.

Ultrasound imaging presents many positive attributes, including safety, real-time imaging, universal accessibility, and cost. However, inherent difficulties in discrimination between soft tissues and tumors prompted development of stabilized microbubble contrast agents. This presents the opportunity to develop agents in which drug is entrapped in the microbubble shell. We describe preparation and characterization of theranostic poly(lactide) (PLA) and pegylated PLA (PEG-PLA) shelled microbubbles that entrap gemcitabine, a commonly used drug for pancreatic cancer (PDAC). Entrapping 6 wt% gemcitabine did not significantly affect drug activity, microbubble morphology, or ultrasound contrast activity compared with unmodified microbubbles. In vitro microbubble concentrations yielding ≥ 500nM entrapped gemcitabine were needed for complete cell death in MIA PaCa-2 PDAC drug sensitivity assays, compared with 62.5 nM free gemcitabine. In vivo administration of gemcitabine-loaded microbubbles to xenograft MIA PaCa-2 PDAC tumors in athymic mice was well tolerated and provided substantial tumoral image enhancement before and after destructive ultrasound pulses. However, no significant differences in tumor growth were observed among treatment groups, in keeping with the in vitro observation that much higher doses of gemcitabine are required to mirror free gemcitabine activity. STATEMENT OF SIGNIFICANCE: The preliminary results shown here are encouraging and support further investigation into increased gemcitabine loading. Encapsulation of gemcitabine within polylactic acid (PLA) microbubbles does not damage its activity towards pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC) cells. Excellent imaging and evidence of penetration into the highly desmoplastic PDAC tumors is demonstrated. Microbubble destruction was confirmed in vivo, showing that elevated mechanical index shatters the microbubbles for enhanced delivery. The potential to slow PDAC growth in vivo is shown, but higher gemcitabine concentrations are required. Current efforts are directed at increasing drug loading by inclusion of drug-carrying nanoparticles for effective in vivo treatment

Negative feedback loop of bone resorption by NFATc1-dependent induction of Cadm1.

Trimethylation of histone H3 lysine 4 and lysine 27 (H3K4me3 and H3K27me3) at gene promoter regions critically regulates gene expression. Key developmental genes tend to exhibit changes in histone modification patterns from the H3K4me3/H3K27me3 bivalent pattern to the H3K4me3 monovalent pattern. Using comprehensive chromatin immunoprecipitation followed by sequencing in bone marrow-derived macrophages (BMMs) and mature osteoclasts, we found that cell surface adhesion molecule 1 (Cadm1) is a direct target of nuclear factor of activated T cells 1 (NFATc1) and exhibits a bivalent histone pattern in BMMs and a monovalent pattern in osteoclasts. Cadm1 expression was upregulated in BMMs by receptor activator of nuclear factor kappa B ligand (RANKL), and blocked by a calcineurin/NFATc1 inhibitor, FK506. Cadm1-deficient mice exhibited significantly reduced bone mass compared with wild-type mice, which was due to the increased osteoclast differentiation, survival and bone-resorbing activity in Cadm1-deficient osteoclasts. These results suggest that Cadm1 is a direct target of NFATc1, which is induced by RANKL through epigenetic modification, and regulates osteoclastic bone resorption in a negative feedback manner