Hippo and PI5P4K signaling intersect to control the transcriptional activation of YAP.

Phosphoinositides are essential signaling molecules. The PI5P4K family of phosphoinositide kinases and their substrates and products, PI5P and PI4,5P2, respectively, are emerging as intracellular metabolic and stress sensors. We performed an unbiased screen to investigate the signals that these kinases relay and the specific upstream regulators controlling this signaling node. We found that the core Hippo pathway kinases MST1/2 phosphorylated PI5P4Ks and inhibited their signaling in vitro and in cells. We further showed that PI5P4K activity regulated several Hippo- and YAP-related phenotypes, specifically decreasing the interaction between the key Hippo proteins MOB1 and LATS and stimulating the YAP-mediated genetic program governing epithelial-to-mesenchymal transition. Mechanistically, we showed that PI5P interacted with MOB1 and enhanced its interaction with LATS, thereby providing a signaling connection between the Hippo pathway and PI5P4Ks. These findings reveal how these two important evolutionarily conserved signaling pathways are integrated to regulate metazoan development and human disease.

Enhanced SREBP2-driven cholesterol biosynthesis by PKCλ/ι deficiency in intestinal epithelial cells promotes aggressive serrated tumorigenesis.

The metabolic and signaling pathways regulating aggressive mesenchymal colorectal cancer (CRC) initiation and progression through the serrated route are largely unknown. Although relatively well characterized as BRAF mutant cancers, their poor response to current targeted therapy, difficult preneoplastic detection, and challenging endoscopic resection make the identification of their metabolic requirements a priority. Here, we demonstrate that the phosphorylation of SCAP by the atypical PKC (aPKC), PKCλ/ι promotes its degradation and inhibits the processing and activation of SREBP2, the master regulator of cholesterol biosynthesis. We show that the upregulation of SREBP2 and cholesterol by reduced aPKC levels is essential for controlling metaplasia and generating the most aggressive cell subpopulation in serrated tumors in mice and humans. Since these alterations are also detected prior to neoplastic transformation, together with the sensitivity of these tumors to cholesterol metabolism inhibitors, our data indicate that targeting cholesterol biosynthesis is a potential mechanism for serrated chemoprevention.

PI5P4Kα supports prostate cancer metabolism and exposes a survival vulnerability during androgen receptor inhibition.

Phosphatidylinositol (PI)regulating enzymes are frequently altered in cancer and have become a focus for drug development. Here, we explore the phosphatidylinositol-5-phosphate 4-kinases (PI5P4K), a family of lipid kinases that regulate pools of intracellular PI, and demonstrate that the PI5P4Kα isoform influences androgen receptor (AR) signaling, which supports prostate cancer (PCa) cell survival. The regulation of PI becomes increasingly important in the setting of metabolic stress adaptation of PCa during androgen deprivation (AD), as we show that AD influences PI abundance and enhances intracellular pools of PI-4,5-P2. We suggest that this PI5P4Kα-AR relationship is mitigated through mTORC1 dysregulation and show that PI5P4Kα colocalizes to the lysosome, the intracellular site of mTORC1 complex activation. Notably, this relationship becomes prominent in mouse prostate tissue following surgical castration. Finally, multiple PCa cell models demonstrate marked survival vulnerability following stable PI5P4Kα inhibition. These results nominate PI5P4Kα as a target to disrupt PCa metabolic adaptation to castrate resistance.

Expanding role of PI5P4Ks in cancer: A promising druggable target.

Cancer cells are challenged by a myriad of microenvironmental stresses, and it is their ability to efficiently adapt to the constantly changing nutrient, energy, oxidative, and/or immune landscape that allows them to survive and proliferate. Such adaptations, however, result in distinct vulnerabilities that are attractive therapeutic targets. Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are a family of druggable stress-regulated phosphoinositide kinases that become conditionally essential as a metabolic adaptation, paving the way to targeting cancer cell dependencies. Further, PI5P4Ks have a synthetic lethal interaction with the tumor suppressor p53, the loss of which is one of the most prevalent genetic drivers of malignant transformation. PI5P4K's emergence as a crucial axis in the expanding landscape of phosphoinositide signaling in cancer has already stimulated the development of specific inhibitors. Thus, a better understanding of the biology of the PI5P4Ks will allow for targeted and effective therapeutic interventions. Here, we attempt to summarize the mounting roles of the PI5P4Ks in cancer, including evidence that targeting them is a therapeutic vulnerability and promising next-in-line treatment for multiple cancer subtypes.

PI5P4Ks drive metabolic homeostasis through peroxisome-mitochondria interplay.

PI5P4Ks are a class of phosphoinositide kinases that phosphorylate PI-5-P to PI-4,5-P2. Distinct localization of phosphoinositides is fundamental for a multitude of cellular functions. Here, we identify a role for peroxisomal PI-4,5-P2 generated by the PI5P4Ks in maintaining energy balance. We demonstrate that PI-4,5-P2 regulates peroxisomal fatty acid oxidation by mediating trafficking of lipid droplets to peroxisomes, which is essential for sustaining mitochondrial metabolism. Using fluorescent-tagged lipids and metabolite tracing, we show that loss of the PI5P4Ks significantly impairs lipid uptake and ß-oxidation in the mitochondria. Further, loss of PI5P4Ks results in dramatic alterations in mitochondrial structural and functional integrity, which under nutrient deprivation is further exacerbated, causing cell death. Notably, inhibition of the PI5P4Ks in cancer cells and mouse tumor models leads to decreased cell viability and tumor growth, respectively. Together, these studies reveal an unexplored role for PI5P4Ks in preserving metabolic homeostasis, which is necessary for tumorigenesis.

JAK Inhibitors Suppress Cancer Cachexia-Associated Anorexia and Adipose Wasting in Mice.

BACKGROUND: Cachexia, a syndrome of muscle atrophy, adipose loss, and anorexia, is associated with reduced survival in cancer patients. The colon adenocarcinoma C26c20 cell line secretes the cytokine leukemia inhibitory factor (LIF) which induces cachexia. We characterized how LIF promotes cachexia-associated weight loss and anorexia in mice through JAK-dependent changes in adipose and hypothalamic tissues. METHODS: Cachexia was induced in vivo with the heterotopic allotransplanted administration of C26c20 colon adenocarcinoma cells or the intraperitoneal administration of recombinant LIF in the absence or presence of JAK inhibitors. Blood, adipose, and hypothalamic tissues were collected and processed for cyto/adipokine ELISAs, immunoblot analysis, and quantitative RT-PCR. Cachexia-associated lipolysis was induced in vitro by stimulating differentiated adipocytes with recombinant LIF or IL-6 in the absence or presence of lipase or JAK inhibitors. These adipocytes were processed for glycerol release into the media, immunoblot analysis, and RT-PCR. RESULTS: Tumor-secreted LIF induced changes in adipose tissue expression and serum levels of IL-6 and leptin in a JAK-dependent manner influencing cachexia-associated adipose wasting and anorexia. We identified two JAK inhibitors that block IL-6 family-mediated adipocyte lipolysis and IL-6 induction using an in vitro cachexia lipolysis assay. JAK inhibitors administered to the in vivo C26c20 cancer cachexia mouse models led to 1) a decrease in STAT3 phosphorylation in hypothalamic and adipose tissues, 2) a reverse in the cachexia serum cyto/adipokine signature, 3) a delay in cancer cachexia-associated anorexia and adipose loss, and 4) an improvement in overall survival. CONCLUSIONS: JAK inhibitors suppress LIF-associated adipose loss and anorexia in both in vitro and in vivo models of cancer cachexia.

Cachexia-associated adipose loss induced by tumor-secreted leukemia inhibitory factor is counterbalanced by decreased leptin.

Cachexia syndrome consists of adipose and muscle loss, often despite normal food intake. We hypothesized that cachexia-associated adipose wasting is driven in part by tumor humoral factors that induce adipocyte lipolysis. We developed an assay to purify secreted factors from a cachexia-inducing colon cancer line that increases lipolysis in adipocytes and identified leukemia inhibitory factor (LIF) by mass spectrometry. Recombinant LIF induced lipolysis in vitro. Peripheral LIF administered to mice caused >50% loss of adipose tissue and >10% reduction in body weight despite only transient hypophagia due to decreasing leptin. LIF-injected mice lacking leptin (ob/ob) resulted in persistent hypophagia and loss of adipose tissue and body weight. LIF's peripheral role of initiating lipolysis in adipose loss was confirmed in pair-fed ob/ob mouse studies. Our studies demonstrate that (a) LIF is a tumor-secreted factor that promotes cachexia-like adipose loss when administered peripherally, (b) LIF directly induces adipocyte lipolysis, (c) LIF has the ability to sustain adipose and body weight loss through an equal combination of peripheral and central contributions, and (d) LIF's central effect is counterbalanced by decreased leptin signaling, providing insight into cachexia's wasting, despite normophagia.

Temporal control of bidirectional lipid-droplet motion in Drosophila depends on the ratio of kinesin-1 and its co-factor Halo.

During bidirectional transport, individual cargoes move continuously back and forth along microtubule tracks, yet the cargo population overall displays directed net transport. How such transport is controlled temporally is not well understood. We analyzed this issue for bidirectionally moving lipid droplets in Drosophila embryos, a system in which net transport direction is developmentally controlled. By quantifying how the droplet distribution changes as embryos develop, we characterize temporal transitions in net droplet transport and identify the crucial contribution of the previously identified, but poorly characterized, transacting regulator Halo. In particular, we find that Halo is transiently expressed; rising and falling Halo levels control the switches in global distribution. Rising Halo levels have to pass a threshold before net plus-end transport is initiated. This threshold level depends on the amount of the motor kinesin-1: the more kinesin-1 is present, the more Halo is needed before net plus-end transport commences. Because Halo and kinesin-1 are present in common protein complexes, we propose that Halo acts as a rate-limiting co-factor of kinesin-1.

Ocimum Sanctum seeds, a natural superdisintegrant: formulation and evaluation of fast melt tablets of nimesulide.

BACKGROUND: Fast melt tablets, also known as fast dissolving tablets, disintegrate instantaneously within the mouth and thus can be consumed without water. The present study was aimed to formulate fast melt tablets of nimesulide by using Ocimum Sanctum seeds as a natural tablet superdisintegrant. MATERIAL AND METHODS: Powdered Ocimum seeds were characterized for powder flow properties (bulk density, tapped density, Carr's consolidation index, Hausner ratio, angle of repose), swelling index, viscosity, pH, loss on drying and microbial load. The prepared tablets were evaluated for different tablet parametric tests, wetting time, water absorption ratio, effective pore radius, porosity, packing fraction, in vitro and in vivo disintegration time, in-vitro dissolution and stability studies. RESULTS: The swelling index was evaluated to be 1600. An appreciable effect of the natural material was seen on tablet hardness and friability. The water absorption ratio increased from 56.15 +/- 0.85 to 80.76 +/- 0.70 (A1-A4). Water uptake coupled natural polymer swelling could be the most probable mechanism for concentration dependent reduction in disintegration time by the Ocimum Sanctum seeds. Porosity of the formulated tablets was found to increase from batch A1-A4. The in vitro disintegration results were in line with in vivo disintegration results. The f2 values (in comparison with Nimulid MD) of 95.90 and 93.65 were obtained with A3 and A4 batches respectively. CONCLUSION: It could be concluded that Ocimum Sanctum seeds could be used as a natural superdisintegrant in the formulation of fast melt tablets.

Current clinical coverage of Radiation Therapy Oncology Group-defined target volumes for postmastectomy radiation therapy.

PURPOSE: The Radiation Therapy Oncology Group (RTOG) has published consensus guidelines for contouring relevant anatomy for postmastectomy radiation therapy (RT). How these contours relate to current treatment practices is unknown. We analyzed the dose-volume histograms (DVHs) for these contours using current clinical practice at University of Texas MD Anderson Cancer Center and compared them with the proposed treatment plans to treat RTOG-defined targets to full dose. METHODS AND MATERIALS: We retrospectively analyzed treatment plans for 20 consecutive women treated with postmastectomy RT for which the treatment targets were the chest wall (CW), level III axilla (Ax3), supraclavicular (SCV), and internal mammary (IM) nodes. The RTOG consensus definitions were used to contour the following anatomic structures: CW; level I, II, and III axillary nodes (Ax1, Ax2, Ax3); SCV; IM; and heart (H). DVHs for these contours and the ipsilateral lung were generated from clinically designed treatment that had actually been delivered to each patient. For comparison regarding dose to normal tissue, new treatment plans were generated with the goal of covering 95% of the anatomic contours to 45 Gy. RESULTS: The prescribed dose was 50 Gy in each case. The mean percent of volumes that received 45 Gy (V45) for the RTOG guideline-based contours were CW 74%, Ax1 84%, Ax2 88%, Ax3 96%, SCV 84%, and IM 80%. Mean heart V10 values were 11% for treatment of left-sided tumors and 6% for right-sided tumors. Mean ipsilateral lung V20 values were 28% for left-sided tumors and 34% for right-sided tumors. For the contour-based plans, mean V45 values were CW 94%, Ax1 95%, Ax2 97%, Ax3 98%, SCV 98%, and IM 85%. Mean heart V10 values were 14% for treatment of left-sided tumors and 12% for right-sided tumors. Mean ipsilateral lung V20 values were 32% for left-sided tumors and 45% for right-sided tumors. CONCLUSIONS: Clinically derived treatment plans, which have proven efficacy and are the current standard, cover 74% to 96% of the anatomy-based RTOG consensus volumes to the prescription dose. This discrepancy should be considered if treatment planning protocol guidelines are designed to incorporate these new definitions.

Formulation and evaluation of controlled release matrix mucoadhesive tablets of domperidone using Salvia plebeian gum.

The aim of study was to prepare controlled release matrix mucoadhesive tablets of domperidone using Salvia plebeian gum as natural polymer. Tablets were formulated by direct compression technology employing the natural polymer in different concentrations (5, 10, 15 and 20% w/w). The prepared batches were evaluated for drug assay, diameter, thickness, hardness and tensile strength, swelling index, mucoadhesive strength (using texture analyzer) and subjected to in vitro drug release studies. Real-time stability studies were also conducted on prepared batches. In vitro drug release data were fitted in various release kinetic models for studying the mechanism of drug release. Tensile strength was found to increase from 0.808 ± 0.098 to 1.527 ± 0.10 mN/cm(2) and mucoadhesive strength increased from 13.673 ± 1.542 to 40.378 ± 2.345 N, with an increase in the polymer concentration from 5 to 20% (A1 to A4). Swelling index was reported to increase with both increase in the concentration of gum and the time duration. The in vitro drug release decreased from 97.76 to 83.4% (A1 to A4) with the increase in polymer concentration. The drug release from the matrix tablets was found to follow zero-order and Higuchi models, indicating the matrix-forming potential of natural polymer. The value of n was found to be between 0.5221 and 0.8992, indicating the involvement of more than one drug release mechanism from the formulation and possibly the combination of both diffusion and erosion. These research findings clearly indicate the potential of S. plebeian gum to be used as binder, release retardant and mucoadhesive natural material in tablet formulations.

Taste masked microspheres of ofloxacin: formulation and evaluation of orodispersible tablets.

Ofloxacin is a synthetic chemotherapeutic antibiotic used for treatment of a variety of bacterial infections, but therapy suffers from low patients' compliance due to its unpleasant taste. This study was aimed to develop taste masked microspheres of ofloxacin using Eudragit and to prepare orodispersible tablets of the formulated microspheres using natural superdisintegrant. Taste masking Eudragit E100 microspheres were prepared by solvent evaporation technique with an entrapment efficiency ranging from 69.54 ± 1.98 to 86.52 ± 2.25%. DSC revealed no interaction between the drug and polymer. Microspheres prepared at a drug/polymer ratio of 1:4 and 1:5 revealed sufficient flow properties and better taste masking as compared to other ratios. Drug loaded microspheres were formulated as orodispersible tablets using locust bean gum as a natural superdisintegrant offering the advatages of biocompatibility and biodegrad-ability. The wetting time, water absorption ratio and in-vitro disintegration time of the tablets were found to range between 19 ± 2 to 10 ± 3 seconds, 59.11 ± 0.65 to 85.76 ± 0.96 and 22 ± 2 to 10 ± 2 seconds, respectively. The in-vitro ofloxacin release was about 97.25% within 2h. The results obtained from the study suggested the use of eudragit polymer for preparing ofloxacin loaded microspheres with an aim to mask the bitter taste of the drug and furthermore orodispersible tablets could be formulated using locust bean gum as a natural superdisintegrant.

Formulation and evaluation of mucoadhesive matrix tablets of taro gum: optimization using response surface methodology.

The present study was aimed to formulate and evaluate oral controlled release mucoadhesive matrix tablets of taro gum incorporating domperidone as model drug. Tablets were prepared by direct compression and were evaluated for bioadhesive strength and in vitro dissolution parameters. A central composite design for 2 factors, at 3 levels each, was employed to evaluate the effect of criti cal formulation variables, namely the amount of taro gum (X1) and PVP K 30 (X2), on mucoadhesive strength, tensile strength, release exponent (n) and t50 (time for 50% drug release). The mucoadhesive detachment force (evaluated using texture analyzer) was found to be 18.266, 54.684 and 65.904 N for A4, A5 and A6 batches of the formulated tablets. The polynomial equation indicates that taro gum has dominating effect on mucoadhesive strength and both X1 and X2 have almost equal and comparable effect on tensile strength. The drug release follows first order kinetics (release of drug depends on remaining concentration of drug) and shows best linearity (r2 = 0.983) with higuchi model. The release exponent (n) lies between 0.339 and 0.543 indicating drug release from the matrix tablets may be fickian or non fickian (anamolous) depending upon the concentration of natural polymer. T50 was 58, 140 and 220 minutes for A7, A8 and A9 batches showing overriding potential of taro gum but still the effect of PVP K 30 is noteworthy. PVP K 30 has indirect effect on all the factors by increasing tensile strength and making the tablet firm and intact.

Locust bean gum as superdisintegrant--formulation and evaluation of nimesulide orodispersible tablets.

Orodispersible tablets disperse instantaneously in the mouth so that they can be swallowed without the aid of water. The aim of the present study was to formulate nimesulide orodispersible tablets using locust bean gum as a natural superdisintegrant. The gum was evaluated for powder flow properties, swelling index and loss on drying. Excellent powder flow properties were observed, swelling index was found to be 20 which indicated appreciable capability of locust bean gum to be used as superdisintegrant. The prepared tablets were evaluated against standard superdisintegrant i.e. cross-carmellose sodium. Disintegration time of tablets containing 10 % locust bean gum was found to be 13 seconds. The prepared batches were also evaluated for wetting time, water absorption ratio, effective pore radius, porosity, in vitro and in vivo disintegration time, in vitro release and stability studies. Wetting time was found to reduce from 19 +/- 2 to 11 +/- 3 sec (A1-A4) and 51 +/- 2 to 36 +/- 3 sec (B1-B4). Effective pore radius and porosity were found to be increase with increase in polymer concentration. The superdisintegrant property of locust bean gum may be due to concentration dependent wicking action leading to formation of porous structure which disintegrates the tablet within seconds. In-vivo results were complementary to in-vitro disintegration time results. The in-vitro release studies were compared against marketed nimesulide fast dissolving tablets (Nimulid MD). Stability studies showed that there was no significant change in hardness, friability, tensile strength and assay of the prepared formulations. The f2 values (in comparison with Nimulid MD) of 92.27 and 98.19 were obtained with A3 and A4 batches respectively.

Lallemantia reylenne seeds as superdisintegrant: Formulation and evaluation of nimesulide orodispersible tablets.

AIM: Orodispersible tablets also known as fast dissolving tablets disintegrate instantaneously within the mouth and thus can be consumed without water. The present study was aimed to formulate orodispersible tablets of nimesulide by using Lallemantia reylenne seeds as natural superdisintegrant. MATERIALS AND METHODS: Powdered lallemantia seeds were characterized for powder flow properties (bulk density, tapped density, carr's consolidation index, hausner ratio, angle of repose), swelling index, viscosity, pH, and loss on drying. The prepared tablets were evaluated for different tablet parametric tests, wetting time, water absorption ratio, effective pore radius, porosity, packing fraction, in vitro and in vivo disintegration time, in vitro dissolution and stability studies. RESULTS AND DISCUSSION: Increase in Lallementia reylenne concentration had an appreciable effect on tablet hardness and friability which clearly indicated binding potential of the seeds. Water absorption ratio increased with increase in Lallemantia reylenne concentration from batch A1 to A4. Water uptake coupled natural polymer swelling could be the most probable mechanism for concentration dependent reduction in disintegration time by the Lallemantia reylenne seeds. Porosity of the formulated tablets was found to increase from batch A1-A4. The in vitro disintegration results were in line with in vivo disintegration results. CONCLUSION: It could be concluded that Lallemantia reylenne seeds could be used as natural superdisintegrant in the formulation of orodispersible tablets.