Uptake of endocytic markers into mitotic yeast cells

Abstract[3H]α-Factor and Lucifer yellow were used to measure receptor mediated and fluid-phase endocytosis in the yeast Saccharomyces cerevisiae, arrested in mitosis by depolymerization of the microtubules or due to a mutation preventing nuclear division (cdc16. Both processes continued at roughly the same level as during interphase. This shows that in yeast endocytosis is not interrupted during mitosis like in mammalian cells

Androgen receptor-dependent and -independent mechanisms driving prostate cancer progression: Opportunities for therapeutic targeting from multiple angles.

Despite aggressive treatment for localized cancer, prostate cancer (PC) remains a leading cause of cancer-related death for American men due to a subset of patients progressing to lethal and incurable metastatic castrate-resistant prostate cancer (CRPC). Organ-confined PC is treated by surgery or radiation with or without androgen deprivation therapy (ADT), while options for locally advanced and disseminated PC include radiation combined with ADT, or systemic treatments including chemotherapy. Progression to CRPC results from failure of ADT, which targets the androgen receptor (AR) signaling axis and inhibits AR-driven proliferation and survival pathways. The exact mechanisms underlying the transition from androgen-dependent PC to CRPC remain incompletely understood. Reactivation of AR has been shown to occur in CRPC despite depletion of circulating androgens by ADT. At the same time, the presence of AR-negative cell populations in CRPC has also been identified. While AR signaling has been proposed as the primary driver of CRPC, AR-independent signaling pathways may represent additional mechanisms underlying CRPC progression. Identification of new therapeutic strategies to target both AR-positive and AR-negative PC cell populations and, thereby, AR-driven as well as non-AR-driven PC cell growth and survival mechanisms would provide a two-pronged approach to eliminate CRPC cells with potential for synthetic lethality. In this review, we provide an overview of AR-dependent and AR-independent molecular mechanisms which drive CRPC, with special emphasis on the role of the Jak2-Stat5a/b signaling pathway in promoting castrate-resistant growth of PC through both AR-dependent and AR-independent mechanisms

Exosome-mediated Transfer of αvβ3 Integrin from Tumorigenic to Nontumorigenic Cells Promotes a Migratory Phenotype.

The αvβ3 integrin is known to be highly upregulated during cancer progression and promotes a migratory and metastatic phenotype in many types of tumors. We hypothesized that the αvβ3 integrin is transferred through exosomes and, upon transfer, has the ability to support functional aberrations in recipient cells. Here, for the first time, it is demonstrated that αvβ3 is present in exosomes released from metastatic PC3 and CWR22Pc prostate cancer cells. Exosomal β3 is transferred as a protein from donor to nontumorigenic and tumorigenic cells as β3 protein or mRNA levels remain unaffected upon transcription or translation inhibition in recipient cells. Furthermore, it is shown that upon exosome uptake, de novo expression of an αvβ3 increases adhesion and migration of recipient cells on an αvβ3 ligand, vitronectin. To evaluate the relevance of these findings, exosomes were purified from the blood of TRAMP mice carrying tumors where the expression of αvβ3 is found higher than in exosomes from wild-type mice. In addition, it is demonstrated that αvβ3 is coexpressed with synaptophysin, a biomarker for aggressive neuroendocrine prostate cancer. IMPLICATIONS: Overall this study reveals that the αvβ3 integrin is transferred from tumorigenic to nontumorigenic cells via exosomes, and its de novo expression in recipient cells promotes cell migration on its ligand. The increased expression of αvβ3 in exosomes from mice bearing tumors points to its clinical relevance and potential use as a biomarker. Mol Cancer Res; 14(11); 1136-46. ©2016 AACR

Transcription factor signal transducer and activator of transcription 5 promotes growth of human prostate cancer cells in vivo

Purpose: Stat5a/b is the key mediator of prolactin (Prl) effects in prostate cancer cells via activation of Jak2. Prl is locally produced growth factor in human prostate cancer. Prl protein expression and constitutive activation of Stat5a/b are associated with high histological grade of clinical prostate cancer. Moreover, activation of Stat5a/b in primary prostate cancer predicts early disease recurrence. Here, we inhibited Stat5a/b by several different methodological approaches. Our goal was to establish a proof-of-principle that Stat5a/b is critical for prostate cancer cell viability in vitro and for prostate tumor growth in vivo. Experimental Design: We inhibited Stat5a/b protein expression by antisense oligonucleotides or RNA interference and transcriptional activity of Stat5a/b by adenoviral expression of a dominant-negative mutant of Stat5a/b in prostate cancer cells in culture. Moreover, Stat5a/b activity was suppressed in human prostate cancer xenograft tumors in nude mice. Stat5a/b regulation of BclXL and Cyclin-D1 protein levels was demonstrated by antisense suppression of Stat5a/b protein expression followed by Western blotting. Results and Conclusions: We show here that inhibition of Stat5a/b by antisense oligonuleotides, RNA interference, or adenoviral expression of DNStat5a/b all effectively kill prostate cancer cells. Moreover, we demonstrate that Stat5a/b is critical for human prostate cancer xenograft growth in nude mice. Stat5a/b effects on the viability of on prostate cancer cells involve Stat5a/b-regulation of BclXL and Cyclin-D1 protein levels, but not the expression or activation of Stat3. This work establishes Stat5a/b as a therapeutic target protein for prostate cancer. Pharmacological inhibition of Stat5a/b in prostate cancer can be achieved by small-molecule inhibitors of transactivation, dimerization or DNA-binding of Stat5a/b

Signal transducer and activator of transcription-5 mediates neuronal apoptosis induced by inhibition of Rac GTPase activity.

In several neuronal cell types, the small GTPase Rac is essential for survival. We have shown previously that the Rho family GTPase inhibitor Clostridium difficile toxin B (ToxB) induces apoptosis in primary rat cerebellar granule neurons (CGNs) principally via inhibition of Rac GTPase function. In the present study, incubation with ToxB activated a proapoptotic Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, and a pan-JAK inhibitor protected CGNs from Rac inhibition. STAT1 expression was induced by ToxB; however, CGNs from STAT1 knock-out mice succumbed to ToxB-induced apoptosis as readily as wild-type CGNs. STAT3 displayed enhanced tyrosine phosphorylation following treatment with ToxB, and a reputed inhibitor of STAT3, cucurbitacin (JSI-124), reduced CGN apoptosis. Unexpectedly, JSI-124 failed to block STAT3 phosphorylation, and CGNs were not protected from ToxB by other known STAT3 inhibitors. In contrast, STAT5A tyrosine phosphorylation induced by ToxB was suppressed by JSI-124. In addition, roscovitine similarly inhibited STAT5A phosphorylation and protected CGNs from ToxB-induced apoptosis. Consistent with these results, adenoviral infection with a dominant negative STAT5 mutant, but not wild-type STAT5, significantly decreased ToxB-induced apoptosis of CGNs. Finally, chromatin immunoprecipitation with a STAT5 antibody revealed increased STAT5 binding to the promoter region of prosurvival Bcl-xL. STAT5 was recruited to the Bcl-xL promoter region in a ToxB-dependent manner, and this DNA binding preceded Bcl-xL down-regulation, suggesting transcriptional repression. These data indicate that a novel JAK/STAT5 proapoptotic pathway significantly contributes to neuronal apoptosis induced by the inhibition of Rac GTPase

Pharmacologic suppression of JAK1/2 by JAK1/2 inhibitor AZD1480 potently inhibits IL-6-induced experimental prostate cancer metastases formation.

Metastatic prostate cancer is lethal and lacks effective strategies for prevention or treatment, requiring novel therapeutic approaches. Interleukin-6 (IL-6) is a cytokine that has been linked with prostate cancer pathogenesis by multiple studies. However, the direct functional roles of IL-6 in prostate cancer growth and progression have been unclear. In the present study, we show that IL-6 is produced in distant metastases of clinical prostate cancers. IL-6-activated signaling pathways in prostate cancer cells induced a robust 7-fold increase in metastases formation in nude mice. We further show that IL-6 promoted migratory prostate cancer cell phenotype, including increased prostate cancer cell migration, microtubule reorganization, and heterotypic adhesion of prostate cancer cells to endothelial cells. IL-6-driven metastasis was predominantly mediated by Stat3 and to lesser extent by ERK1/2. Most importantly, pharmacologic inhibition of Jak1/2 by AZD1480 suppressed IL-6-induced signaling, migratory prostate cancer cell phenotypes, and metastatic dissemination of prostate cancer in vivo in nude mice. In conclusion, we demonstrate that the cytokine IL-6 directly promotes prostate cancer metastasis in vitro and in vivo via Jak-Stat3 signaling pathway, and that IL-6-driven metastasis can be effectively suppressed by pharmacologic targeting of Jak1/2 using Jak1/2 inhibitor AZD1480. Our results therefore provide a strong rationale for further development of Jak1/2 inhibitors as therapy for metastatic prostate cancer

Peptidylarginine Deiminase 3 (PAD3) Is Upregulated by Prolactin Stimulation of CID-9 Cells and Expressed in the Lactating Mouse Mammary Gland

Peptidylarginine deiminases (PADs) post-translationally convert arginine into neutral citrulline residues. Our past work shows that PADs are expressed in the canine and murine mammary glands; however, the mechanisms regulating PAD expression and the function of citrullination in the normal mammary gland are unclear. Therefore, the first objective herein was to investigate regulation of PAD expression in mammary epithelial cells. We first examined PAD levels in CID-9 cells, which were derived from the mammary gland of mid-pregnant mice. PAD3 expression is significantly higher than all other PAD isoforms and mediates protein citrullination in CID-9 cells. We next hypothesized that prolactin regulates PAD3 expression. To test this, CID-9 cells were stimulated with 5 mug/mL of prolactin for 48 hours which significantly increases PAD3 mRNA and protein expression. Use of a JAK2 inhibitor and a dominant negative (DN)-STAT5 adenovirus indicate that prolactin stimulation of PAD3 expression is mediated by the JAK2/STAT5 signaling pathway in CID-9 cells. In addition, the human PAD3 gene promoter is prolactin responsive in CID-9 cells. Our second objective was to investigate the expression and activity of PAD3 in the lactating mouse mammary gland. PAD3 expression in the mammary gland is highest on lactation day 9 and coincident with citrullinated proteins such as histones. Use of the PAD3 specific inhibitor, Cl4-amidine, indicates that PAD3, in part, can citrullinate proteins in L9 mammary glands. Collectively, our results show that upregulation of PAD3 is mediated by prolactin induction of the JAK2/STAT5 signaling pathway, and that PAD3 appears to citrullinate proteins during lactation

Autocrine prolactin promotes prostate cancer cell growth via Janus kinase-2-signal transducer and activator of transcription-5a/b signaling pathway.

The molecular mechanisms that promote progression of localized prostate cancer to hormone-refractory and disseminated disease are poorly understood. Prolactin (Prl) is a local growth factor produced in high-grade prostate cancer, and exogenously added Prl in tissue or explant cultures of normal and malignant prostate is a strong mitogen and survival factor for prostate epithelium. The key signaling proteins that mediate the biological effects of Prl in prostate cancer are Signal Transducer and Activator of Transcription (Stat)-5a/5b via activation of Janus kinase-2. Importantly, inhibition of Stat5a/b in prostate cancer cells induces apoptotic death. Using a specific Prl receptor antagonist (Delta1-9G129R-hPRL), we demonstrate here for the first time that autocrine Prl in androgen-independent human prostate cancer cells promotes cell viability via Stat5 signaling pathway. Furthermore, we examined a unique clinical material of human hormone refractory prostate cancers and metastases and show that autocrine Prl is expressed in 54% of hormone-refractory clinical human prostate cancers and 62% prostate cancer metastases. Finally, we demonstrate that autocrine Prl is expressed from both the proximal and distal promoters of the Prl gene in clinical human prostate cancers and in vivo and in vitro human prostate cancer models, independently of pituitary transcription factor-1 (Pit-1). Collectively, the data provide novel evidence for the concept that autocrine Prl signaling pathway is involved in growth of hormone-refractory and metastatic prostate cancer. The study also provides support for the use of Prl receptor antagonists or other therapeutic strategies to block the Prl-Janus kinase-2-Stat5 signaling pathway in advanced prostate cancer

Obesity accelerates epigenetic aging in middle-aged but not in elderly individuals

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