Bone Microenvironment Changes in Latexin Expression Promote Chemoresistance.

Although docetaxel is the standard of care for advanced prostate cancer, most patients develop resistance to docetaxel. Therefore, elucidating the mechanism that underlies resistance to docetaxel is critical to enhance therapeutic intervention. Mining cDNA microarray from the PC-3 prostate cancer cell line and its docetaxel-resistant derivative (PC3-TxR) revealed decreased latexin (LXN) expression in the resistant cells. LXN expression was inversely correlated with taxane resistance in a panel of prostate cancer cell lines. LXN knockdown conferred docetaxel resistance to prostate cancer cells in vitro and in vivo, whereas LXN overexpression reduced docetaxel resistance in several prostate cancer cell lines. A mouse model of prostate cancer demonstrated that prostate cancer cells developed resistance to docetaxel in the bone microenvironment, but not the soft tissue microenvironment. This was associated with decreased LXN expression in prostate cancer cells in the bone microenvironment compared with the soft tissue microenvironment. It was identified that bone stromal cells decreased LXN expression through methylation and induced chemoresistance in prostate cancer in vitro These findings reveal that a subset of prostate cancer develops docetaxel resistance through loss of LXN expression associated with methylation and that the bone microenvironment promotes this drug resistance phenotype.Implications: This study suggests that the LXN pathway should be further explored as a viable target for preventing or reversing taxane resistance in prostate cancer. Mol Cancer Res; 15(4); 457-66. ©2017 AACR.

Mechanisms of Metastatic Tumor Dormancy.

Tumor metastasis can occur years after an apparent cure due to a phenomenon known as metastatic tumor dormancy; in which tumor masses or individual tumor cells are growth restricted for extended periods of time. This period of dormancy is induced and maintained by several mechanisms, including: (1) Tumor microenvironment factors such as cytokine expression, immunosurveillance and angiogenesis; (2) Metastasis suppressor gene activity; and (3) Cancer therapeutics. Disseminated tumor cells (DTC) are the key cells that result in dormant tumors. However, many challenges exist towards isolating DTCs for mechanistic studies. The main DTC that may represent the dormant cell is the cancer stem cells (CSC) as they have a slow proliferation rate. In addition to limited knowledge regarding induction of tumor dormancy, there are large gaps in knowledge regarding how tumors escape from dormancy. Emerging research into cancer stem cells, immunotherapy, and metastasis suppressor genes, may lead to new approaches for targeted anti-metastatic therapy to prevent dormancy escape. Overall, an enhanced understanding of tumor dormancy is critical for better targeting and treatment of patients to prevent cancer recurrence.

A role for phospholipase D3 in myotube formation.

Phospholipase D3 (PLD3) is a non-classical, poorly characterized member of the PLD superfamily of signaling enzymes. PLD3 is a type II glycoprotein associated with the endoplasmic reticulum, is expressed in a wide range of tissues and cells, and undergoes dramatic upregulation in neurons and muscle cells during differentiation. Using an in vitro skeletal muscle differentiation system, we define the ER-tethering mechanism and report that increased PLD3 expression enhances myotube formation, whereas a putatively dominant-negative PLD3 mutant isoform reduces myotube formation. ER stress, which also enhances myotube formation, is shown here to increase PLD3 expression levels. PLD3 protein was observed to localize to a restricted set of subcellular membrane sites in myotubes that may derive from or constitute a subdomain of the endoplasmic reticulum. These findings suggest that PLD3 plays a role in myogenesis during myotube formation, potentially in the events surrounding ER reorganization.

Phosphatidylinositol-4-phosphate-5-kinase alpha deficiency alters dynamics of glucose-stimulated insulin release to improve glucohomeostasis and decrease obesity in mice.

OBJECTIVE: Phosphatidylinositol-4-phosphate-5-kinase (PI4P5K) has been proposed to facilitate regulated exocytosis and specifically insulin secretion by generating phosphatidylinositol-4,5-bisphosphate (PIP(2)). We sought to examine the role of the α isoform of PI4P5K in glucohomeostasis and insulin secretion. RESEARCH DESIGN AND METHODS: The response of PI4P5Kα(-/-) mice to glucose challenge and a type 2-like diabetes-inducing high-fat diet was examined in vivo. Glucose-stimulated responses and PI4P5Kα(-/-) pancreatic islets and ß-cells were characterized in culture. RESULTS: We show that PI4P5Kα(-/-) mice exhibit increased first-phase insulin release and improved glucose clearance, and resist high-fat diet-induced development of type 2-like diabetes and obesity. PI4P5Kα(-/-) pancreatic islets cultured in vitro exhibited decreased numbers of insulin granules docked at the plasma membrane and released less insulin under quiescent conditions, but then secreted similar amounts of insulin on glucose stimulation. Stimulation-dependent PIP(2) depletion occurred on the plasma membrane of the PI4P5Kα(-/-) pancreatic ß-cells, accompanied by a near-total loss of cortical F-actin, which was already decreased in the PI4P5Kα(-/-) ß-cells under resting conditions. CONCLUSIONS: Our findings suggest that PI4P5Kα plays a complex role in restricting insulin release from pancreatic ß-cells through helping to maintain plasma membrane PIP(2) levels and integrity of the actin cytoskeleton under both basal and stimulatory conditions. The increased first-phase glucose-stimulated release of insulin observed on the normal diet may underlie the partial protection against the elevated serum glucose and obesity seen in type 2 diabetes-like model systems.

Monitoring insulin-stimulated production of signaling lipids at the plasma membrane.

Lipid second messengers play important roles in many cell signaling cascades. Lipid signaling molecules allow for high specificity, rapid transduction, and rapid reversibility of localized stimulation events. Fluorescent sensors capable of detecting individual signaling lipids enable their production and degradation to be followed, revealing the nature and dynamics of signaling pathways. The following sections outline a method for using lipid sensors to monitor the production of signaling lipids on the plasma membrane of C2C12 myotubes in response to insulin signaling.

Design of expression vectors for RNA interference based on miRNAs and RNA splicing.

RNA interference (RNAi) mediates sequence-specific post-transcriptional gene silencing in many eukaryotes and is used for reverse genetic studies and therapeutics. RNAi is triggered by double-stranded small interfering RNAs (siRNAs), which can be processed from small hairpin RNAs generated from an expression vector. In some recently described vectors, the siRNAs are expressed from synthetic stem-loop precursors of microRNAs (miRNAs) driven by polymerase II promoters. We have designed new RNAi vectors, designated pSM155 and pSM30, that take into consideration miRNA processing and RNA splicing by placing the miRNA-based artificial miRNA expression cassettes inside of synthetic introns. Like the original miRNA vectors, we show that the pSM155 and pSM30 constructs efficiently down-regulate expression of firefly luciferase and an endogenous gene, phospholipase D2. Moreover, the expression of a coexpressed fluorescent marker is substantially improved by this new design. Another improvement of these new vectors is incorporation of two inverted BsmBI sites placed internal to the arms of the new miRNA-based vectors, so oligos used for cloning are shorter and the cost is reduced. These RNAi vectors thus provide new tools for gene suppression.

The usher N terminus is the initial targeting site for chaperone-subunit complexes and participates in subsequent pilus biogenesis events.

Pilus biogenesis on the surface of uropathogenic Escherichia coli requires the chaperone/usher pathway, a terminal branch of the general secretory pathway. In this pathway, periplasmic chaperone-subunit complexes target an outer membrane (OM) usher for subunit assembly into pili and secretion to the cell surface. The molecular mechanisms of protein secretion across the OM are not well understood. Mutagenesis of the P pilus usher PapC and the type 1 pilus usher FimD was undertaken to elucidate the initial stages of pilus biogenesis at the OM. Deletion of residues 2 to 11 of the mature PapC N terminus abolished the targeting of the usher by chaperone-subunit complexes and rendered PapC nonfunctional for pilus biogenesis. Similarly, an intact FimD N terminus was required for chaperone-subunit binding and pilus biogenesis. Analysis of PapC-FimD chimeras and N-terminal fragments of PapC localized the chaperone-subunit targeting domain to the first 124 residues of PapC. Single alanine substitution mutations were made in this domain that blocked pilus biogenesis but did not affect targeting of chaperone-subunit complexes. Thus, the usher N terminus does not function simply as a static binding site for chaperone-subunit complexes but also participates in subsequent pilus assembly events.