► Carboxylated agarose was prepared by TEMPO oxidation system and characterized. ► Ag-g-DA was obtained through EDC condensation reaction and characterized. ► Carboxylated agarose and Ag-g-DA showed ...no cytotoxicity and enhanced cell adhesion.
In order to improve bioactivity of agarose, we modified agarose by carboxylation and grafting dopamine. Under alkaline condition, carboxylated agarose was prepared using 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO) oxidation system by oxidizing C6 hydroxyl on d-galactose ring into carboxyl group, and the maximum value of the degree of carboxylation reached 30%. With the increase of the amount of oxidant, the molecular weight of the carboxylated agarose decreased to 4kDa by gel permeation chromatography (GPC) measure. Carboxylated agarose reacted with dopamine through EDC condensation reaction to obtain agarose grafting dopamine (Ag-g-DA), and the grafting rate of dopamine was determined to be 9.3% by UV spectroscopy at 280nm. The structures of these modified agaroses were determined by FT-IR and 13C NMR. Both carboxylated agarose and Ag-g-DA showed no cytotoxicity and promoted cell-adhesiveness.
Sustained reliance on androgen receptor (AR) after failure of AR-targeting androgen deprivation therapy (ADT) prevents effective treatment of castration-recurrent (CR) prostate cancer (CaP). ...Interfering with the molecular machinery by which AR drives CaP progression may be an alternative therapeutic strategy but its feasibility remains to be tested. Here, we explore targeting the mechanism by which AR, via RhoA, conveys androgen-responsiveness to serum response factor (SRF), which controls aggressive CaP behavior and is maintained in CR-CaP. Following a siRNA screen and candidate gene approach, RNA-Seq studies confirmed that the RhoA effector Protein Kinase N1 (PKN1) transduces androgen-responsiveness to SRF. Androgen treatment induced SRF-PKN1 interaction, and PKN1 knockdown or overexpression severely impaired or stimulated, respectively, androgen regulation of SRF target genes. PKN1 overexpression occurred during clinical CR-CaP progression, and hastened CaP growth and shortened CR-CaP survival in orthotopic CaP xenografts. PKN1's effects on SRF relied on its kinase domain. The multikinase inhibitor lestaurtinib inhibited PKN1 action and preferentially affected androgen regulation of SRF over direct AR target genes. In a CR-CaP patient-derived xenograft, expression of SRF target genes was maintained while AR target gene expression declined and proliferative gene expression increased. PKN1 inhibition decreased viability of CaP cells before and after ADT. In patient-derived CaP explants, lestaurtinib increased AR target gene expression but did not significantly alter SRF target gene or proliferative gene expression. These results provide proof-of-principle for selective forms of ADT that preferentially target different fractions of AR's transcriptional output to inhibit CaP growth.
Although tremendous efforts have been made on targeted drug delivery systems, current therapy outcomes still suffer from low circulating time and limited targeting efficiency. The integration of ...cell‐mediated drug delivery and theranostic nanomedicine can potentially improve cancer management in both therapeutic and diagnostic applications. By taking advantage of innate immune cell's ability to target tumor cells, the authors develop a novel drug delivery system by using macrophages as both nanoparticle (NP) carriers and navigators to achieve cancer‐specific drug delivery. Theranostic NPs are fabricated from a unique polymer, biodegradable photoluminescent poly (lactic acid) (BPLP‐PLA), which possesses strong fluorescence, biodegradability, and cytocompatibility. In order to minimize the toxicity of cancer drugs to immune cells and other healthy cells, an anti‐BRAF V600E mutant melanoma specific drug (PLX4032) is loaded into BPLP‐PLA nanoparticles. Muramyl tripeptide is also conjugated onto the nanoparticles to improve the nanoparticle loading efficiency. The resulting nanoparticles are internalized within macrophages, which are tracked via the intrinsic fluorescence of BPLP‐PLA. Macrophages carrying nanoparticles deliver drugs to melanoma cells via cell–cell binding. Pharmacological studies also indicate that the PLX4032 loaded nanoparticles effectively kill melanoma cells. The “self‐powered” immune cell‐mediated drug delivery system demonstrates a potentially significant advancement in targeted theranostic cancer nanotechnologies.
By taking advantage of the innate immune cell's ability to target tumor cells, a novel drug delivery system is developed by using THP‐1 cells as both nanoparticle‐carriers and navigators to achieve cancer‐specific drug delivery. The “self‐powered” immune cell‐mediated theranostic biodegradable photoluminescent poly (lactic acid) nanoparticle‐based drug delivery system represents a potentially significant advancement in targeted theranostic cancer nanotechnologies.
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Novel citric acid based photoluminescent dyes and biodegradable polymers are synthesized via a facile “one-pot” reaction. A comprehensive understanding of the fluorescence mechanisms ...of the resulting citric acid-based fluorophores is reported. Two distinct types of fluorophores are identified: a thiozolopyridine family with high quantum yield, long lifetime, and exceptional photostability, and a dioxopyridine family with relatively lower quantum yield, multiple lifetimes, and solvent-dependent band shifting behavior. Applications in molecular labeling and cell imaging were demonstrated. The above discoveries contribute to the field of fluorescence chemistry and have laid a solid foundation for further development of new fluorophores and materials that show promise in a diversity of fluorescence-based applications.
Photoluminescent materials are pivotal for fluorescence based imaging, labeling and sensing applications. Understanding their fluorescence mechanism is challenging and imperative. We develop a new class of citric acid-derived fluorescent materials in forms of polymers and small molecular dyes by a one-step solvent free reaction. We discovered two different classes of citric acid-derived fluorophores. A two-ring thiozolopyridine structure demonstrates strong fluorescence and exceptional resistance to photo-bleaching. A one-ring dioxopyridine exhibits relative weak fluorescence but with intriguing excitation and solvent-dependent emission wavelength shifting. Our methodology of synthesizing citric acid-derived fluorophores and the understanding on their luminescence are instrumental to the design and production of a large number of new photoluminescent materials for biological and biomedical applications.
Abstract
Ligand-activated androgen receptor (AR) drives prostate cancer (CaP) growth. Heterogeneity in AR action exists and AR often remains active when therapies targeting its ligand-activation have ...failed. Understanding the mechanisms by which AR mediates CaP progression may lead to new treatments. Previously, we identified a mechanism in which AR imparts androgen-responsiveness to the transcription factor Serum Response Factor (SRF). SRF-dependent AR action is enriched in CaP, correlates with CaP recurrence and is maintained in late-stage CaP. SRF binds constitutively at its target genes and becomes transcriptionally active via upstream signaling cascades or SRF cofactors. Our prior work showed that RhoA signaling transduces androgen regulation to a significant fraction, but not all, SRF target genes. Here, we isolate the homeodomain protein Nkx2-5 as a novel regulator of androgen-responsive SRF action. Nkx2-5 co-operates with SRF in transcriptional control of heart development. In CaP, Nkx2-5 promoter hypermethylation is a biomarker that distinguishes CaP from benign prostate, yet consequences of differential Nkx2-5 expression on CaP are unknown. In the CaP cell models LNCaP, C4-2 and VCaP, siRNA-mediated loss of Nkx2-5 increased basal and androgen-dependent expression of exogenously and endogenously expressed SRF target genes, and these effects were reversed after silencing of SRF. Conversely, overexpression of Nkx2-5 decreased androgen-responsiveness of SRF target genes. Co-IP and ChIP assays indicated androgen-stimulated SRF-Nkx2-5 interaction in CaP cell nuclei. Loss of Nkx2-5 resulted in distinct morphological changes in CaP cells, which depended entirely on SRF. In rhodamine phalloidin staining assays, loss of Nkx2-5 increased significantly (p<0.01) cell length, width and circumference, and the number of cell protrusions in cultures with and without androgen stimulation. Changes in cell shape were accompanied by modest decreases in CaP cell proliferation and migration, but significant (p<0.05) decreases in CaP cell-matrix adhesion. Nkx2-5 silencing increased the size of the cell nucleus and led to multilobed and grooved nuclei and enlarged nucleoli. These nuclear shape changes were not associated with alterations in DNA ploidy, but were associated with changed heterochromatin organization as reflected in a reduction in heterochromatin protein 1 immunostaining and nuclear content. Such Nkx2-5-dependent changes in nuclear shape and size and heterochromatin organization occurred also in non-malignant prostate epithelial RWPE2 cells. This work uncovered a novel role for Nkx2-5 in the transition from benign prostate to CaP, showing that Nkx2-5 regulates SRF transcriptional activity in CaP cells and demonstrating that differential Nkx2-5 expression alters cell-matrix adhesion and nuclear morphology, both processes that are associated with aggressive CaP progression.
Citation Format: Salma Ben-Salem, Dhirodatta Senapati, Giridhar Mudduluru, Yixue Su, Varadha Balaji Venkadakrishnan, Hannelore V. Heemers. The cardiac transcription factor Nkx2-5 contributes to SRF-dependent AR action in prostate cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-211.
Prostate cancer remains the second leading cause of cancer death in men in Western cultures. A deeper understanding of the mechanisms by which prostate cancer cells divide to support tumor growth ...could help devise strategies to overcome treatment resistance and improve survival. Here, we identified that the mitotic AGC family protein kinase citron kinase (CIT) is a pivotal regulator of prostate cancer growth that mediates prostate cancer cell interphase progression. Increased CIT expression correlated with prostate cancer growth induction and aggressive prostate cancer progression, and CIT was overexpressed in prostate cancer compared with benign prostate tissue. CIT overexpression was controlled by an E2F2-Skp2-p27 signaling axis and conferred resistance to androgen-targeted treatment strategies. The effects of CIT relied entirely on its kinase activity. Conversely, CIT silencing inhibited the growth of cell lines and xenografts representing different stages of prostate cancer progression and treatment resistance but did not affect benign epithelial prostate cells or nonprostatic normal cells, indicating a potential therapeutic window for CIT inhibition. CIT kinase activity was identified as druggable and was potently inhibited by the multikinase inhibitor OTS-167, which decreased the proliferation of treatment-resistant prostate cancer cells and patient-derived organoids. Isolation of the in vivo CIT substrates identified proteins involved in diverse cellular functions ranging from proliferation to alternative splicing events that are enriched in treatment-resistant prostate cancer. These findings provide insights into the regulation of aggressive prostate cancer cell behavior by CIT and identify CIT as a functionally diverse and druggable driver of prostate cancer progression.
The poorly characterized protein kinase citron kinase is a therapeutic target in prostate cancer that drives tumor growth by regulating diverse substrates, which control several hallmarks of aggressive prostate cancer progression. See related commentary by Mishra et al., p. 4008.
Abstract
Androgen receptor (AR) is the major driver of prostate cancer (CaP) progression. Despite initially inducing remissions, androgen deprivation therapy (ADT) does not cure CaP and the majority ...of CaP that recurs under ADT continues to rely on AR. In a subset of patients, administration of novel, more potent ADT drugs leads to emergence of a neuroendocrine (NE) CaP phenotype that is AR-indifferent and even harder to treat. The molecular mechanisms by which AR regulates cell cycle progression and that can be developed into alternative targets to inhibit CaP growth, overcome acquired resistance to ADT, or prevent NE progression, however, remain largely unknown. Here, we identify citron kinase (CIT), which controls cell division, as a novel drugabble target that acts downstream of AR. In multiple CaP models, CIT protein expression was stimulated selectively by low doses of androgens that promote CaP cell proliferation and decreased by AR silencing or short-term administration of AR-antagonist enzalutamide, confirming its AR dependence. Silencing of CIT significantly reduced CaP cell viability and cell proliferation, delayed cell progression, increased the number of multinucleated cells in ADT-naïve and -recurrent CaP cells, and attenuated CaP growth in xenograft models. Overexpression of CIT, transiently or stably, stimulated CaP cell proliferation in androgen-supplemented conditions and under ADT, and this depended entirely on an intact CIT kinase domain. In 2 independent clinical CaP datasets, CIT mRNA levels increased during CaP progression and higher CIT expression correlated with shorter disease-free and overall survival. Using CaP tissue microarrays that contain more than 200 patient specimens, CIT protein expression was significantly higher in primary CaP when compared to adjacent non-neoplastic prostate tissues and correlated with increasing Gleason scores, validating the relevance of CIT overexpression to CaP aggressiveness. Integrated RNA-Seq, MSigDB and GSEA analyses confirmed that the AR- and CIT-dependent transcriptome preferentially controlled CaP cell cycle progression and proliferation. Mechanistically, the use of inhibitors of gene transcription, mRNA translation and proteasome indicated that the androgen-dependence of CIT expression is regulated at the post-transcriptional level. Specifically, CIT protein expression was controlled by the E2F family of cell cycle regulators, with androgen-responsive E2F2 as the major determinant of CaP CIT expression. Our work, thus, isolated a novel role for the mitotic kinase CIT in AR-dependent CaP cell proliferation and clinical progression and identified CIT’s kinase moiety as a novel target for CaP therapy.
Funding: NIH/NCI, Case Comprehensive Cancer Center Pilot Research Award
Citation Format: Salma Ben-Salem, Salam Bachour, Varadha Balaji Venkadakrishnan, Yixue Su, Eduardo Cortes Gomez, Qiang Hu, Eric Klein, Nicolas Marlo, Cristina Magi-Galluzzi, Liu Song, Hannelore V. Heemers. Prostate cancer progression depends on the activity of the mitotic kinase citron kinase abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 390.
Abstract
A major limitation in the management of castration-recurrent prostate cancer (CR-CaP) is the lack of treatments to inhibit androgen receptor (AR) action that is driving CaP growth when ...androgen-deprivation therapy (ADT) has failed. AR action breaks down in fractions that are regulated differently at the molecular level, control diverse aspects of CaP biology and contribute differentially to CaP progression. Blocking a specific fraction(s) of AR action that mediates CaP progression may serve as alternative treatment strategy, yet evidence for such an approach and drugs that render this approach feasible are not known. Our laboratory has identified a mechanism of AR action in which AR conveys androgen-responsiveness to Serum Response Factor (SRF) target genes via androgen activation of RhoA. Androgen-responsive SRF action mediates cell migration and is associated with aggressive CaP behavior and recurrence. Importantly, control of AR over RhoA-SRF action is maintained in CR-CaP, which renders this signaling a potentially attractive novel therapeutic target. Using an siRNA screen, we identified Protein Kinase N1 (PKN1) as the Rho effector that transduces androgen-responsiveness from RhoA to SRF. In promoter-reporter, qRT-PCR and oligoarray assays, knockdown of PKN1 severely impaired the androgen-regulation of SRF target genes, but affected androgen response of only a few (12 of 452) direct AR target genes. In contrast, transient overexpression of PKN1 preferentially stimulated androgen-responsive SRF target gene expression. Stable overexpression of PKN1 hastened growth and shortened survival in CaP orthotopic xenografts. Site-directed mutagenesis showed that the effect of PKN1 on SRF target genes relied on its kinase activity. Treatment with lestaurtinib, a multikinase inhibitor that is also a potent inhibitor of PKN1, mimicked the effects of PKN1 knockdown on expression of SRF and AR target genes in several CaP cell lines and ex vivo CaP explants. Lestaurtinib also inhibited the proliferation of CaP cells that are either ADT-naïve or -resistant, express only the constitutively active AR variant ARv567es, or are growth-stimulated by the AR-activating ADT drug metabolite 5α-abiraterone. RNA-Seq followed by MSigDB analyses confirmed that lestaurtinib impairs androgen-dependent PKN1 activity and revealed that 100 of the 127 MgSigDB gene sets isolated after PKN1 silencing overlapped with 123 gene sets derived after lestaurtinib treatment. Overlapping gene sets included several related to hormonal carcinogenesis and cancer progression, prostate development, signaling cascades that have been implicated in SRF and RhoA function and/or are relevant to CaP. PKN1 inhibition via lestaurtinib, which is used already to treat human hematologic malignancies and is well tolerated by carcinoma patients, may thus serve as novel alternative treatment strategy to target AR-dependent SRF action in CR-CaP.
Citation Format: Varadha Balaji Venkadarkrishnan, Adam DePriest, Yixue Su, Giridhar Mudduluru, Salma Ben-Salem, Sangeeta Kumari, Qiang Hu, Eduardo Cortes, Scott Dehm, Cristina Magi-Galluzzi, Eric Klein, Nima Sharifi, Song Liu, Hannelore Heemers. Inhibition of Protein Kinase N1 prevents control of AR over SRF action in advanced prostate cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3745.
By taking advantage of the innate ability of the immune cells to target tumor cells, in article number 1603121, Cheng Dong, Jian Yang, and co‐workers develop a selfpowered immune cell‐mediated ...theranostic biodegradable photoluminescent poly (lactic acid) (BPLP‐PLA) nanoparticle‐based drug delivery system for melanoma targeting and drug delivery.
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