Heat shock factor 1 directly regulates transsulfuration pathway to promote prostate cancer proliferation and survival.

There are limited therapeutic options for patients with advanced prostate cancer (PCa). We previously found that heat shock factor 1 (HSF1) expression is increased in PCa and is an actionable target. In this manuscript, we identify that HSF1 regulates the conversion of homocysteine to cystathionine in the transsulfuration pathway by altering levels of cystathionine-ß-synthase (CBS). We find that HSF1 directly binds the CBS gene and upregulates CBS mRNA levels. Targeting CBS decreases PCa growth and induces tumor cell death while benign prostate cells are largely unaffected. Combined inhibition of HSF1 and CBS results in more pronounced inhibition of PCa cell proliferation and reduction of transsulfuration pathway metabolites. Combination of HSF1 and CBS knockout decreases tumor size for a small cell PCa xenograft mouse model. Our study thus provides new insights into the molecular mechanism of HSF1 function and an effective therapeutic strategy against advanced PCa.

Targeting glutamine dependence with DRP-104 inhibits proliferation and tumor growth of castration-resistant prostate cancer.

BACKGROUND: Prostate cancer (PCa) continues to be one of the leading causes of cancer deaths in men. While androgen deprivation therapy is initially effective, castration-resistant PCa (CRPC) often recurs and has limited treatment options. Our previous study identified glutamine metabolism to be critical for CRPC growth. The glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) blocks both carbon and nitrogen pathways but has dose-limiting toxicity. The prodrug DRP-104 is expected to be preferentially converted to DON in tumor cells to inhibit glutamine utilization with minimal toxicity. However, CRPC cells' susceptibility to DRP-104 remains unclear. METHODS: Human PCa cell lines (LNCaP, LAPC4, C4-2/MDVR, PC-3, 22RV1, NCI-H660) were treated with DRP-104, and effects on proliferation and cell death were assessed. Unbiased metabolic profiling and isotope tracing evaluated the effects of DRP-104 on glutamine pathways. Efficacy of DRP-104 in vivo was evaluated in a mouse xenograft model of neuroendocrine PCa, NCI-H660. RESULTS: DRP-104 inhibited proliferation and induced apoptosis in CRPC cell lines. Metabolite profiling showed decreases in the tricarboxylic acid cycle and nucleotide synthesis metabolites. Glutamine isotope tracing confirmed the blockade of both carbon pathway and nitrogen pathways. DRP-104 treated CRPC cells were rescued by the addition of nucleosides. DRP-104 inhibited neuroendocrine PCa xenograft growth without detectable toxicity. CONCLUSIONS: The prodrug DRP-104 blocks glutamine carbon and nitrogen utilization, thereby inhibiting CRPC growth and inducing apoptosis. Targeting glutamine metabolism pathways with DRP-104 represents a promising therapeutic strategy for CRPC.

The effects of glycemic index on prostate cancer progression in a xenograft mouse model.

BACKGROUND: Previously, we found low-carbohydrate diets slowed prostate cancer (PC) growth and increased survival vs. a Western diet in mice, by inhibiting the insulin/IGF-1 axis. Thus, we tested whether modifying carbohydrate quality to lower glycemic index (GI) without changing quantity results in similar benefits as with reduced quantity. METHODS: Male SCID mice injected with LAPC-4 cells were single-housed and randomized when their tumors reached 200 mm3 on average to a LoGI (48% carbohydrate kcal, from Hylon-VII) or HiGI Western diet (48% carbohydrate kcal, from sucrose). Body weight and tumor volume were measured weekly. Body composition was assessed 35 days after randomization. Blood glucose and serum insulin, IGF-1 and IGFBP3 were measured at study end when tumor volumes reached 800 mm3. We analyzed gene expression of mice tumors by RNA-sequencing and human tumors using the Prostate Cancer Transcriptome Atlas. RESULTS: There were no significant differences in tumor volume (P > 0.05), tumor proliferation (P = 0.29), and overall survival (P = 0.15) between groups. At 35 days after randomization, the LoGI group had 30% lower body fat (P = 0.007) despite similar body weight (P = 0.58). At sacrifice, LoGI mice had smaller livers (P < 0.001) and lower glucose (P = 0.15), insulin (P = 0.11), IGF-1 (P = 0.07) and IGF-1:IGFBP3 ratio (P = 0.05), and higher IGFBP3 (P = 0.09) vs. HiGI, although none of these metabolic differences reached statistical significance. We observed differential gene expression and pathway enrichment in mice tumors by diet. The most upregulated and downregulated gene in the LoGI group showed expression patterns more closely resembling expression in human benign prostate tissue vs. PC. CONCLUSIONS: In this single mouse xenograft model, consuming a low GI diet did not delay PC growth or survival vs. a high GI diet despite suggestions of decreased activation of the insulin/IGF-1 pathway. These data suggest that improving carbohydrate quality alone while consuming a high carbohydrate diet may not effectively slow PC growth.

Targeting the Divergent Roles of STK3 Inhibits Breast Cancer Cell Growth and Opposes Doxorubicin-Induced Cardiotoxicity In Vitro.

Breast cancer (BCa) is the most prevalent type of cancer in women. Several therapies used in the treatment of breast cancer are associated with clinically important rates of cardiovascular toxicity during or after treatment exposure, including anthracyclines. There is a need for new BCa therapeutics and treatments that mitigate chemotherapy-induced cardiotoxicity in BCa. In this study, we examine the effects of Serine/Threonine Kinase 3 (STK3) inhibition in the context of BCa therapy and cardioprotection from doxorubicin. STK3 (also known as MST2) is a key member of the Hippo Tumor-Suppressor Pathway, which regulates cell growth and proliferation by inhibiting YAP/TAZ co-transcription factors. Canonically, STK3 should restrict BCa growth; however, we observed that STK3 is amplified in BCa and associated with worse patient outcomes, suggesting a noncanonical pro-tumorigenic role. We found BCa cell lines have varying dependence on STK3. SUM52PE cells had the highest expression and dependence on STK3 in genetic and pharmacological assays. MCF-7 and MDA-MB-231 were less sensitive to STK3 targeting in standard proliferation assays, but were STK3 dependent in colony formation and matrigel invasion assays. In contrast, STK3 inhibition mitigated the toxic effects of doxorubicin in H9C2 rat cardiomyocytes by increasing YAP expression. Importantly, STK3 inhibition in BCa cells did not interfere with the therapeutic effects of doxorubicin. Our studies highlight STK3 is a potential molecular target for BCa with dual therapeutic effects: suppression of BCa growth and progression, and chemoprotection in cardiomyocytes.

MESH1 knockdown triggers proliferation arrest through TAZ repression.

All organisms are constantly exposed to various stresses, necessitating adaptive strategies for survival. In bacteria, the main stress-coping mechanism is the stringent response triggered by the accumulation of "alarmone" (p)ppGpp to arrest proliferation and reprogram transcriptome. While mammalian genomes encode MESH1-the homolog of the (p)ppGpp hydrolase SpoT, current knowledge about its function remains limited. We found MESH1 expression tended to be higher in tumors and associated with poor patient outcomes. Consistently, MESH1 knockdown robustly inhibited proliferation, depleted dNTPs, reduced tumor sphere formation, and retarded xenograft growth. These antitumor phenotypes associated with MESH1 knockdown were accompanied by a significantly altered transcriptome, including the repressed expression of TAZ, a HIPPO coactivator, and proliferative gene. Importantly, TAZ restoration mitigated many anti-growth phenotypes of MESH1 knockdown, including proliferation arrest, reduced sphere formation, tumor growth inhibition, dNTP depletion, and transcriptional changes. Furthermore, TAZ repression was associated with the histone hypo-acetylation at TAZ regulatory loci due to the induction of epigenetic repressors HDAC5 and AHRR. Together, MESH1 knockdown in human cells altered the genome-wide transcriptional patterns and arrested proliferation that mimicked the bacterial stringent response through the epigenetic repression of TAZ expression.

Non-canonical role of Hippo tumor suppressor serine/threonine kinase 3 STK3 in prostate cancer.

Serine/threonine kinase 3 (STK3) is an essential member of the highly conserved Hippo tumor suppressor pathway that regulates Yes-associated protein 1 (YAP1) and TAZ. STK3 and its paralog STK4 initiate a phosphorylation cascade that regulates YAP1/TAZ inhibition and degradation, which is important for regulated cell growth and organ size. Deregulation of this pathway leads to hyperactivation of YAP1 in various cancers. Counter to the canonical tumor suppression role of STK3, we report that in the context of prostate cancer (PC), STK3 has a pro-tumorigenic role. Our investigation started with the observation that STK3, but not STK4, is frequently amplified in PC. Additionally, high STK3 expression is associated with decreased overall survival and positively correlates with androgen receptor (AR) activity in metastatic castrate-resistant PC. XMU-MP-1, an STK3/4 inhibitor, slowed cell proliferation, spheroid growth, and Matrigel invasion in multiple models. Genetic depletion of STK3 decreased proliferation in several PC cell lines. In a syngeneic allograft model, STK3 loss slowed tumor growth kinetics in vivo, and biochemical analysis suggests a mitotic growth arrest phenotype. To further probe the role of STK3 in PC, we identified and validated a new set of selective STK3 inhibitors, with enhanced kinase selectivity relative to XMU-MP-1, that inhibited tumor spheroid growth and invasion. Consistent with the canonical role, inhibition of STK3 induced cardiomyocyte growth and had chemoprotective effects. Our results indicate that STK3 has a non-canonical role in PC progression and that inhibition of STK3 may have a therapeutic potential for PC that merits further investigation.

NUAK family kinase 2 is a novel therapeutic target for prostate cancer.

Current advancements in prostate cancer (PC) therapies have been successful in slowing PC progression and increasing life expectancy; however, there is still no curative treatment for advanced metastatic castration resistant PC (mCRPC). Most treatment options target the androgen receptor, to which many PCs eventually develop resistance. Thus, there is a dire need to identify and validate new molecular targets for treating PC. We found NUAK family kinase 2 (NUAK2) expression is elevated in PC and mCRPC versus normal tissue, and expression correlates with an increased risk of metastasis. Given this observation and because NUAK2, as a kinase, is actionable, we evaluated the potential of NUAK2 as a molecular target for PC. NUAK2 is a stress response kinase that also plays a role in activation of the YAP cotranscriptional oncogene. Combining pharmacological and genetic methods for modulating NUAK2, we found that targeting NUAK2 in vitro leads to reduction in proliferation, three-dimensional tumor spheroid growth, and matrigel invasion of PC cells. Differential gene expression analysis of PC cells treated NUAK2 small molecule inhibitor HTH-02-006 demonstrated that NUAK2 inhibition results in downregulation of E2F, EMT, and MYC hallmark gene sets after NUAK2 inhibition. In a syngeneic allograft model and in radical prostatectomy patient derived explants, NUAK2 inhibition slowed tumor growth and proliferation rates. Mechanistically, HTH-02-006 treatment led to inactivation of YAP and the downregulation of NUAK2 and MYC protein levels. Our results suggest that NUAK2 represents a novel actionable molecular target for PC that warrants further exploration.

Patrones de distribución y estructura de la vegetación en el gradiente de humedales costeros El Castaño, Chiapas, México

Introducción: Las características de los humedales costeros son resultado de las interacciones hidrogeomorfológicas entre el continente y el océano, que causan un gradiente ambiental, que resulta en diferentes tipos de vegetación como manglares, popales, tulares, selvas y palmares inundables. Objetivo: Caracterizar las variables del hidroperiodo y fisicoquímicas del agua y suelo para determinar la relación que existe en el patrón de distribución de la vegetación en el Sistema de Humedales El Castaño (SHC). Metodología: Se establecieron 11 unidades de muestreo (UM) permanentes por estrato definidos: cinco en el manglar, dos en selvas inundables, dos en tular y dos en pastizal inundable. De mayo 2016 a octubre 2017 se caracterizó la vegetación y se muestreó mensualmente los niveles de inundación y parámetros fisicoquímicos del agua (superficial, intersticial y subterránea): salinidad, conductividad y pH; y el suelo: densidad aparente, porcentaje de humedad y potencial redox. Resultados: El manglar es el más cercano al mar, tiene la menor diversidad (H:1.66) y especies registradas (14), está dominado por Laguncularia racemosa y Rhizophora mangle y tiene los valores más altos de salinidad intersticial y subterránea, mayores a 10.8 ups, se mantiene inundado de 4 a 12 meses, su potencial redox es de 14.57 mV. Seguido está el manglar, tierra adentro, se ubican los remanentes de la selva inundable, (H:2.18 y 18 especies), dominada por Pachiraaquatica, la salinidad intersticial y subterránea de 4.95 ups, permanece inundada de 0 a 6 meses y el potencial redox es de 119.07 mV. El tular, después de la selva, (H:1.92 y 16 especies), dominado por Typha domingensis, salinidad intersticial y subterránea de 6.1 ups, el tiempo de inundación es de 5 a 8 meses y potencial redox es de 125.9 mV. El pastizal inundable, con menor influencia marina, es un humedal herbáceo modificado para uso ganadero, presentó los valores más altos de diversidad (H:3.44 y 50 especies), Paspalum conjugatum es la especie dominante, la salinidad intersticial y subterránea es menor a 0.5 ups, se mantiene inundado de 5 a 9 meses y el potencial redox es de 151.23 mV. Conclusiones: En cada tipo de vegetación, la estructura, composición y diversidad es diferente, con un alto recambio de especies que indica un gradiente definido por la salinidad. La vegetación en el SHC sigue los patrones de organización típica de los humedales costeros tropicales, manglares, selvas inundables y humedales herbáceos, en este caso los tulares y pastizales inundables. El factor que define la distribución de la vegetación, es salinidad y el gradiente que se observa está en función de la dinámica hidrológica que depende de entradas de agua marina y de la bajada de agua dulce del interior del continente.

Introduction: The characteristics of coastal wetlands are the result of hydrogeomorphological interactions between the continent and the ocean, which cause an environmental gradient, hat results in different vegetation types such as mangroves, freshwater marshes, swamp forests and palm swamps. Objective: To characterize the hydroperiod and physicochemical variables of water and soil and their effect on the distribution of vegetation in the Sistema de Humedales El Castaño. Methods: A total of 11 permanent sampling units (UM) were established by defined strata: five in the mangrove, two in swamp forest, two in freshwater marshes and two in the flooded pasture. From May 2016 to October 2017 the vegetation was characterized and the water levels and physicochemical parameters (superficial, interstitial and groundwater) were sampled monthly for: salinity, and pH; and the soil for: bulk density, humidity percentage, and redox potential. Results: Mangroves are the closest to the sea, have the lowest diversity (H: 1.66) and species richness (14), they are dominated by Laguncularia racemosa and Rhizophora mangle, have the highest values of interstitial and groundwater salinity, (> 10.8 ups), remain flooded for 4 to 12 months per year, and have a redox potential of 14.57 mV. Immediately, inland, there are remnants of the swamp forests (H: 2.18 and 18 species), dominated by Pachira aquatica, with 5 ups interstitial and groundwater salinity, flooded from 0 to 6 months per year, with a redox potential of 119.07 mV. These forests are followed inland by freshwater marshes (H: 1.92 and 16 species), dominated by Typha domingensis with 6.1 ups interstitial and groundwater salinity, flooded for 5 to 8 months per year and a redox potential of 125.9 mV. Finally, furthest inland is the flooded pasture, a modified herbaceous wetland for cattle grazing (H: 3.44 and 50 species) dominated by Paspalum conjugatum, where interstitial and groundwater salinity is less than 0.5 ups, it stays flooded for 5 to 9 months and the redox potential is 151.23 mV. Conclusions: In each type of vegetation, the structure, composition, and diversity are different, with a high turnover of species that indicates a gradient defined by salinity. The vegetation in the SHC follows the patterns of typical organization of the tropical coastal wetlands, mangroves, swamp forests and herbaceous wetlands, in this case the freshwater marshes and flooded pastures. The factor that define the distribution of the vegetation is the salinity and the gradient that is observed are a function of the hydrological dynamics that depends on the mixing of marine and freshwater.

27-Hydroxycholesterol Impairs Plasma Membrane Lipid Raft Signaling as Evidenced by Inhibition of IL6-JAK-STAT3 Signaling in Prostate Cancer Cells.

We recently reported that restoring the CYP27A1-27hydroxycholesterol axis had antitumor properties. Thus, we sought to determine the mechanism by which 27HC exerts its anti-prostate cancer effects. As cholesterol is a major component of membrane microdomains known as lipid rafts, which localize receptors and facilitate cellular signaling, we hypothesized 27HC would impair lipid rafts, using the IL6-JAK-STAT3 axis as a model given its prominent role in prostate cancer. As revealed by single molecule imaging of DU145 prostate cancer cells, 27HC treatment significantly reduced detected cholesterol density on the plasma membranes. Further, 27HC treatment of constitutively active STAT3 DU145 prostate cancer cells reduced STAT3 activation and slowed tumor growth in vitro and in vivo. 27HC also blocked IL6-mediated STAT3 phosphorylation in nonconstitutively active STAT3 cells. Mechanistically, 27HC reduced STAT3 homodimerization, nuclear translocation, and decreased STAT3 DNA occupancy at target gene promoters. Combined treatment with 27HC and STAT3 targeting molecules had additive and synergistic effects on proliferation and migration, respectively. Hallmark IL6-JAK-STAT gene signatures positively correlated with CYP27A1 gene expression in a large set of human metastatic castrate-resistant prostate cancers and in an aggressive prostate cancer subtype. This suggests STAT3 activation may be a resistance mechanism for aggressive prostate cancers that retain CYP27A1 expression. In summary, our study establishes a key mechanism by which 27HC inhibits prostate cancer by disrupting lipid rafts and blocking STAT3 activation. IMPLICATIONS: Collectively, these data show that modulation of intracellular cholesterol by 27HC can inhibit IL6-JAK-STAT signaling and may synergize with STAT3-targeted compounds.

Serum cholesterol levels and tumor growth in a PTEN-null transgenic mouse model of prostate cancer.

BACKGROUND: Some, but not all, epidemiologic evidence supports a role for cholesterol, the precursor for steroid hormone synthesis, in prostate cancer. Using a PTEN-null transgenic mouse model of prostate cancer, we tested the effect of modifying serum cholesterol levels on prostate tumor development and growth. We hypothesized that serum cholesterol reduction would lower tumor androgens and slow prostate cancer growth. METHODS: PTENloxP/loxP-Cre+ mice consuming ad libitum high fat, high cholesterol diets (40% fat, 1.25% cholesterol) were randomized after weaning to receive the cholesterol uptake inhibitor, ezetimibe (30 mg/kg/day), or no intervention, and sacrificed at 2, 3, or 4 months of age. Serum cholesterol and testosterone were measured by ELISA and intraprostatic androgens by mass spectrometry. Prostate histology was graded, and proliferation and apoptosis in tumor epithelium and stroma was assessed by Ki67 and TUNEL, respectively. RESULTS: Ezetimibe-treated mice had lower serum cholesterol at 4 months (p = 0.031). Serum cholesterol was positively correlated with prostate weight (p = 0.033) and tumor epithelial proliferation (p = 0.069), and negatively correlated with tumor epithelial apoptosis (p = 0.004). Serum cholesterol was unrelated to body weight (p = 0.195). Tumor stromal cell proliferation was reduced in the ezetimibe group (p = 0.010). Increased serum cholesterol at 4 months was associated with elevated intraprostatic DHEA, testosterone, and androstenedione (p = 0.043, p = 0.074, p = 0.031, respectively). However, cholesterol reduction did not significantly affect adenocarcinoma development at 2, 3, or 4 months of age (0, 78, and 100% in ezetimibe-treated vs. 0, 80, and 100% in mice not receiving ezetimibe). CONCLUSIONS: Though serum cholesterol reduction did not significantly affect the rate of adenocarcinoma development in the PTEN-null transgenic mouse model of prostate cancer, it lowered intraprostatic androgens and slowed tumor growth. These findings support a role for serum cholesterol in promoting prostate cancer growth, potentially via enhanced tumor androgen signaling, and may provide new insight into cholesterol-lowering interventions for prostate cancer treatment.

Angiotensin receptor signaling and prostate tumor growth in mice.

OBJECTIVE: The renin-angiotensin system, through its type 1 and type 2 angiotensin receptors (AT1R and AT2R, respectively) may have a role in prostate cancer. The objective of this pilot study was to explore that potential role by determining whether the AT1R blocker, losartan, would reduce the growth of LAPC-4 prostate cancer xenografts in nude mice. We also evaluated the tumor growth effects of using angiotensin II to activate both AT1R and AT2R simultaneously. Our data showed that losartan decreased tumor volumes by 56% versus control. This decrease reached statistical significance at day 54 (p = 0.0014). By day 54, Ki67 was also reduced in the losartan group, though not significantly so (p = 0.077). Losartan had no significant effect on AT1R or AT2R expression. Despite significant increases in both AT1R and AT2R at day 29 (p = 0.043 and 0.038, respectively), the administration of angiotensin II did not result in any significant differences in tumor volumes or ki67 at any time point. These data suggest that selective activation and induction of AT2R coupled with blockade of AT1R may slow prostate cancer growth. Future larger studies are needed to confirm these results.

CYP27A1 Loss Dysregulates Cholesterol Homeostasis in Prostate Cancer.

In this study, we used a bioinformatic approach to identify genes whose expression is dysregulated in human prostate cancers. One of the most dramatically downregulated genes identified encodes CYP27A1, an enzyme involved in regulating cellular cholesterol homeostasis. Importantly, lower CYP27A1 transcript levels were associated with shorter disease-free survival and higher tumor grade. Loss of CYP27A1 in prostate cancer was confirmed at the protein level by immunostaining for CYP27A1 in annotated tissue microarrays. Restoration of CYP27A1 expression in cells where its gene was silenced attenuated their growth in vitro and in tumor xenografts. Studies performed in vitro revealed that treatment of prostate cancer cells with 27-hydroxycholesterol (27HC), an enzymatic product of CYP27A1, reduced cellular cholesterol content in prostate cancer cell lines by inhibiting the activation of sterol regulatory-element binding protein 2 and downregulating low-density lipoprotein receptor expression. Our findings suggest that CYP27A1 is a critical cellular cholesterol sensor in prostate cells and that dysregulation of the CYP27A1/27HC axis contributes significantly to prostate cancer pathogenesis. Cancer Res; 77(7); 1662-73. ©2017 AACR.

Evidence for Feedback Regulation Following Cholesterol Lowering Therapy in a Prostate Cancer Xenograft Model.

BACKGROUND: Epidemiologic data suggest cholesterol-lowering drugs may prevent the progression of prostate cancer, but not the incidence of the disease. However, the association of combination therapy in cholesterol reduction on prostate or any cancer is unclear. In this study, we compared the effects of the cholesterol lowering drugs simvastatin and ezetimibe alone or in combination on the growth of LAPC-4 prostate cancer in vivo xenografts. METHODS: Proliferation assays were conducted by MTS solution and assessed by Student's t-test. 90 male nude mice were placed on a high-cholesterol Western-diet for 7 days then injected subcutaneously with 1 × 105 LAPC-4 cells. Two weeks post-injection, mice were randomized to control, 11 mg/kg/day simvastatin, 30 mg/kg ezetimibe, or the combination and sacrificed 42 days post-randomization. We used a generalized linear model with the predictor variables of treatment, time, and treatment by time (i.e., interaction term) with tumor volume as the outcome variable. Total serum and tumor cholesterol were measured. Tumoral RNA was extracted and cDNA synthesized from 1 ug of total RNA for quantitative real-time PCR. RESULTS: Simvastatin directly reduced in vitro prostate cell proliferation in a dose-dependent, cell line-specific manner, but ezetimibe had no effect. In vivo, low continuous dosing of ezetimibe, delivered by food, or simvastatin, delivered via an osmotic pump had no effect on tumor growth compared to control mice. In contrast, dual treatment of simvastatin and ezetimibe accelerated tumor growth. Ezetimibe significantly lowered serum cholesterol by 15%, while simvastatin had no effect. Ezetimibe treatment resulted in higher tumor cholesterol. A sixfold induction of low density lipoprotein receptor mRNA was observed in ezetimibe and the combination with simvastatin versus control tumors. CONCLUSIONS: Systemic cholesterol lowering by ezetimibe did not slow tumor growth, nor did the cholesterol independent effects of simvastatin and the combined treatment increased tumor growth. Despite lower serum cholesterol, tumors from ezetimibe treated mice had higher levels of cholesterol. This study suggests that induction of low density lipoprotein receptor is a possible mechanism of resistance that prostate tumors use to counteract the therapeutic effects of lowering serum cholesterol. Prostate 77:446-457, 2017. © 2016 Wiley Periodicals, Inc.

A novel mechanism of skin tumor promotion involving interferon-gamma (IFNγ)/signal transducer and activator of transcription-1 (Stat1) signaling.

The current study was designed to explore the role of signal transducer and activator of transcription 1 (Stat1) during tumor promotion using the mouse skin multistage carcinogenesis model. Topical treatment with both 12-O-tetradecanoylphorbol-13-acetate (TPA) and 3-methyl-1,8-dihydroxy-9-anthrone (chrysarobin or CHRY) led to rapid phosphorylation of Stat1 on both tyrosine (Y701) and serine (S727) residues in epidermis. CHRY treatment also led to upregulation of unphosphorylated Stat1 (uStat1) at later time points. CHRY treatment also led to upregulation of interferon regulatory factor 1 (IRF-1) mRNA and protein, which was dependent on Stat1. Further analyses demonstrated that topical treatment with CHRY but not TPA upregulated interferon-gamma (IFNγ) mRNA in the epidermis and that the induction of both IRF-1 and uStat1 was dependent on IFNγ signaling. Stat1 deficient (Stat1(-/-) ) mice were highly resistant to skin tumor promotion by CHRY. In contrast, the tumor response (in terms of both papillomas and squamous cell carcinomas) was similar in Stat1(-/-) mice and wild-type littermates with TPA as the promoter. Maximal induction of both cyclooxygenase-2 and inducible nitric oxide synthase in epidermis following treatment with CHRY was also dependent on the presence of functional Stat1. These studies define a novel mechanism associated with skin tumor promotion by the anthrone class of tumor promoters involving upregulation of IFNγ signaling in the epidermis and downstream signaling through activated (phosphorylated) Stat1, IRF-1 and uStat1.

Constitutive Stat3 activation alters behavior of hair follicle stem and progenitor cell populations.

STATs play crucial roles in a wide variety of biological functions, including development, proliferation, differentiation and migration as well as in cancer development. In the present study, we examined the impact of constitutive activation of Stat3 on behavior of keratinocytes, including keratinocyte stem cells (KSC) in vivo. BK5.Stat3C transgenic (Tg) mice, which express a constitutively active form of Stat3 (Stat3C) in the basal layer of the epidermis and in the bulge region KSCs exhibited a significantly reduced number of CD34+/α6 integrin+ cells compared to non-transgenic (NTg) littermates. There was a concomitant increase in the Lgr-6, Lrig-1, and Sca-1 populations in the Tg mice in contrast to the CD34 and Keratin-15 positive population. In addition, increased expression of c-myc, ß-catenin, and epithelial-mesenchymal transition (EMT)-related genes as well as decreased expression of α6-integrin was observed in the hair follicles of Tg mice. Notably, Sca-1 was found to be a direct transcriptional target of Stat3 in keratinocytes. The current data suggest that elevated Stat3 activity leads to depletion of hair follicle KSCs along with a concomitant increase of stem/progenitor cells above the bulge region. Overall, the current data indicate that Stat3 plays an important role in keratinocyte stem/progenitor cell homeostasis.

Avifauna en sistemas silvopastoriles en el Corredor Biológico Mesoamericano, Tabasco, México / Avifauna in silvopastoral systems in the Mesoamerican Biological Corridor, Tabasco, México

Silvopastoral systems support local ecological and economical features as they enhance conservation of floral and faunal communities. As other animal communities, avifauna may be a good representative of habitat altera- tions, both as the species and functional levels. In order to attend the initiative of Mesoamerican Biological Corridor initiative (CBM) in the state of Tabasco, we studied the diversity of birds in two silvopastoral systems: scattered trees in pastures (ADP), and trees in boundary-hedgerows (AL). For this, we applied the fixed radius counting point method in three priority sites in Tabasco´s CBM during the dry and wet season of 2011, and a total of 56, 60 and 62 points were evaluated in Huimanguillo, Tenosique and Tacotalpa, respectively. We observed 2 084 individuals of 154 species (79-89% of expected diversity) and 36 bird families. We detected 92, 87 and 85 species in Huimanguillo, Tenosique and Tacotalpa, respectively, including 35 protected species, of which 23, 19 and 16 in each locality, respectively. All sites showed high diversity (H´≥3.20), low species dominance (D≥0.08) and high equitability (J≥0.77). Species composition showed differences between sites, being most similar Tacotalpa and Tenosique. Ten species were considered characteristic for sites. Although the silvopastoral system did contain protected species, the low diversity and the early successional character of the arboreal components were not attractive to frugivorous bird species. Diversification with native trees can improve the systems to create a complementary habitat and to increase landscape connectivity. The management of silvopastoral practices on cattle dominated landscapes in Tabasco could improve its ecological quality, and thus achieve the CBM´s objectives of biodiversity conservation combined with human economic activities.

Los sistemas silvopastoriles contienen rasgos ecológicos y económicos que contribuyen con la conservación de comunidades florísticas y faunísticas que en ellas se desarrollan. Entre otras comunidades faunísticas se encuentra la avifauna la cual es un grupo representativo de las alteraciones del habitat, tanto a nivel específico como grupo functional. Con el objetivo de atender la iniciativa de Corredor Biológico Mesoamericano en Tabasco, México, se analizó la diversidad de aves en dos sistemas silvopastoriles: árboles dispersos en potreros (ADP) y árboles en cercos o linderos (AL). Se aplicó el método de punto de conteo de radio fijo en tres sitios prioritarios del CBM de Tabasco, ubicados en los municipios: Huimanguillo, Tacotalpa y Tenosique. Los datos provienen de las estaciones seca y húmeda del 2011. Se registraron 2 084 aves de 154 especies (79-89% del esperado) y 36 familias. Se detectaron 92, 87 y 85 especies, incluyendo 35 protegidas, con 23, 19 y 16 en Huimanguillo, Tacotalpa y Tenosique, respectivamente. Todos los sitios mostraron alta diversidad (H’≥3.20), baja dominancia de especies (D≥0.08) y alta equidad (J≥0.77). La composición de especies mostró diferencias entre sitios, con mayor similitud entre Tacotalpa y Tenosique. Diez especies pueden considerarse características de los sitios. Aunque en los sistemas silvopastoriles se refugian aves protegidas, el componente arbóreo es poco diverso y corresponde a especies de sucesión secundaria temprana, lo que limita a las aves frugívoras y especialistas de bosque. Diversificar estos sistemas con árboles nativos puede mejorar estos hábitats complementarios e incrementar la conectividad del paisaje para cumplir con los objetivos del CBM en la conservación de la biodiversidad y provisión de bienes a las poblaciones humanas.

Stat3 binds to mtDNA and regulates mitochondrial gene expression in keratinocytes.

The nuclear transcription factor signal transducer and activator of transcription 3 (Stat3) has recently been reported to have a localized mitochondrial regulatory function. Current data suggest that mitochondrial Stat3 (mitoStat3) is necessary for maximal mitochondrial activity and for Ras-mediated transformation independent of Stat3 nuclear activity. We have previously shown that Stat3 has a pivotal role in epithelial carcinogenesis. Therefore, the aim of the current study was to determine the role of mitoStat3 in epidermal keratinocytes. Herein, we show that normal and neoplastic keratinocytes contain a pool of mitoStat3. EGF and 12-O-tetradecanoylphorbol-13-acetate induce Stat3 mitochondrial translocation mediated through the phosphorylation of Stat3 at Ser727. In addition, we report that mitoStat3 binds mtDNA and associates with the mitochondrial transcription factor A. Furthermore, Stat3 ablation resulted in an increase of mitochondrial-encoded gene transcripts. An increase in key nuclear-encoded metabolic genes, PGC-1α and NRF-1, was also observed in Stat3-null keratinocytes; however, no changes in nuclear-encoded electron transport chain gene transcripts or mtDNA copy number were observed. Collectively, our findings suggest a heretofore-unreported function for mitoStat3 as a potential mitochondrial transcription factor in keratinocytes. This mitoStat3-mtDNA interaction may represent an alternate signaling pathway that could alter mitochondrial function and biogenesis and have a role in tumorigenesis.

Efecto de microorganismos con potencial probiótico en la calidad del agua y el crecimiento de camarón Litopenaeus vannamei (Decapoda: Penaeidae) en cultivo intensivo / Microorganisms effect with probiotic potential in water quality and growth of the shrimp Litopenaeus vannamei (Decapoda: Penaeidae) in intensive culture

The use of probiotics has gained acceptance in aquaculture, particularly in maintaining water quality and enhancing growth in organisms. This study analyzed the effect of the commercial (EM TM, Japan) natural product composed by (Rhodopseudomonas palustris, Lactobacillus plantarum, Lactobacillus casei and Saccharomyces cerevisiae) added to the water, in order to determine its effect in water quality, sediment and growth of L. vannamei under intensive culture. The evaluation included three treatments with a weekly addition of EM: i) tanks without probiotics (C), ii) tanks with a dose of 4L/ha (EM1) and iii) tanks with a dose of 10L/ha (EM2). The treatment C was carried out three times, while treatments EM1 and EM2 were carried out four times. A total of 4 350 shrimps were measured for total length and weight, to calculate total and porcentual weight gain, daily weight gain, specific growth rate (TCE), and food conversion factor (FCA); besides, the survival rate was estimated. The use of probiotics allowed a shorter harvest time in treatments EM1 (90d) and EM2 (105d) with relation to the treatment C (120d). Treatments EM1 and EM2 were within the recommended intervals for culture, with respect to treatment C. The use of probiotic bacteria significantly regulated pH (EM1, 8.03±0.33; EM2, 7.77±0.22; C, 9.08±0.35) and reduced nitrate concentration (EM1, 0.64±0.25mg/L; EM2, 0.39±0.26mg/L; C, 0.71mg/L). Water pH mostly explained the variance with respect to the treatments. Treatment EM2 presented the greatest removal of organic matter (1.77±0.45%), whereas the contents of extractable phosphorus increased significantly in treatment EM1 with 21.6±7.99mg/kg and in treatment EM2 with 21.6±8.45mg/kg with control relation (14.3±5.47). The shrimp growth was influenced by dissolved oxygen, salinity and pH in the sediment, establishing that salinity was the most important variable in the weight with a negative association. Treatment EM1 recorded an improved TCE (2.69±0.35%/d) and FCA (1.46±0.20) with relation to the control treatment (TCE, 1.88±0.25%/d; FCA, 2.13±0.48). Survival was significantly greater in treatments containing probiotics with 61±8.76% and 60±10.5% for EM1 and EM2, respectively. This study indicated the positive effect obtained with the use of this commercial probiotic, to improve culture conditions and growth parameters in an intensive culture of L. vannamei.

Los probióticos han ganado aceptación en la acuicultura para mantener la calidad del agua y aumentar el crecimiento de los organismos. En este estudio se analizó el efecto de una mezcla comercial de microorganismos eficientes (EM) (Rhodopseudomonas palustris, Lactobacillus plantarum, Lactobacillus casei y Saccharomyces cerevisiae) sobre la calidad del agua, sedimento y el crecimiento en un cultivo intensivo de camarón L. vannamei. La evaluación consistió en tres tratamientos: i) estanques sin EM (C), ii) estanques con dosis de 4L/ha (EM1) y iii) estanques con dosis de 10L/ha (EM2). Los resultados demostraron menor tiempo de cosecha en los tratamientos EM1 (90d) y EM2 (105d). Los tratamientos EM1 y EM2 mantuvieron significativamente regulados los valores del pH (EM1, 8.03±0.33; EM2, 7.77±0.22) y redujeron las concentraciones de nitrato (EM1, 0.64±0.25mg/L; EM2, 0.39±0.26mg/L). El tratamiento EM2 presentó la mayor remoción de materia orgánica (1.77±0.45%). El tratamiento EM1 mejoró la TCE (2.69±0.35%/d) y FCA (1.46±0.20). Los tratamientos EM1 y EM2 presentaron mayor supervivencia con 61±8.76% y 60±10.5%, respectivamente. Este estudio demostró el efecto benéfico del uso de la mezcla comercial en los parámetros ambientales y de crecimiento en un cultivo intensivo de L. vannamei.

Role of stat3 in skin carcinogenesis: insights gained from relevant mouse models.

Signal transducer and activator of transcription 3 (Stat3) is a cytoplasmic protein that is activated in response to cytokines and growth factors and acts as a transcription factor. Stat3 plays critical roles in various biological activities including cell proliferation, migration, and survival. Studies using keratinocyte-specific Stat3-deficient mice have revealed that Stat3 plays an important role in skin homeostasis including keratinocyte migration, wound healing, and hair follicle growth. Use of both constitutive and inducible keratinocyte-specific Stat3-deficient mouse models has demonstrated that Stat3 is required for both the initiation and promotion stages of multistage skin carcinogenesis. Further studies using a transgenic mouse model with a gain of function mutant of Stat3 (Stat3C) expressed in the basal layer of the epidermis revealed a novel role for Stat3 in skin tumor progression. Studies using similar Stat3-deficient and gain-of-function mouse models have indicated its similar roles in ultraviolet B (UVB) radiation-mediated skin carcinogenesis. This paper summarizes the use of these various mouse models for studying the role and underlying mechanisms for the function of Stat3 in skin carcinogenesis. Given its significant role throughout the skin carcinogenesis process, Stat3 is an attractive target for skin cancer prevention and treatment.

Skp2 deficiency inhibits chemical skin tumorigenesis independent of p27(Kip1) accumulation.

S-phase kinase-associated protein 2 (Skp2) functions as the receptor component of the Skp-Cullin-F-box complex and is implicated in the degradation of several cell cycle regulators, such as p21(Cip1), p27(Kip1), p57(Kip2), and cyclin E. Numerous studies in human and experimental tumors have demonstrated low p27(Kip1) levels and elevated Skp2 expression. However, a direct association between the inverse correlation of Skp2 and p27(Kip1) with tumorigenesis has not been demonstrated. Herein, we provide evidence that skin tumorigenesis is inhibited in Skp2(-/-) mice. An analysis of mouse keratinocytes indicates that increased p27(Kip1) levels in Skp2(-/-) epidermis cause reduced cell proliferation that is alleviated in the epidermis from Skp2(-/-)/p27(-/-) compound mice. In contrast, we establish that a p27(Kip1) deficiency does not overturn the reduced skin tumorigenesis experienced by Skp2(-/-) mice. In addition, Skp2(-/-) epidermis exhibits an accumulation of p53-cofactor CBP/p300 that is associated with elevated apoptosis in hair follicles and decreased skin tumorigenesis. We conclude that p27(Kip1) accumulation is responsible for the hypoplasia observed in normal tissues of Skp2(-/-) mice but does not have a preponderant function in reducing skin tumorigenesis.