VGLL1 stabilization of cytoplasmic TAZ promotes EGFR expression and maintains tumor initiating cells in breast cancer independent of TEAD.

Vestigial-like family member 1 (VGLL1) is one of the X-linked genes whose expression is elevated in basal-like breast cancer (BLBC) because of X-chromosome isodisomy. As an approach towards understanding its function, we performed correlation study using transcript data of breast cancer patients from cBioPortal for Cancer Genomics. Our analysis identified EGFR as the most correlated transcript with VGLL1. We demonstrate that VGLL1 promotes EGFR expression and increases the frequency of breast tumor initiating cells (CD44high/+CD24low/-). These findings are crucial because an elevated EGFR expression and high frequency of CD44high/+CD24low/- cells are defining features of BLBC, and we provide a new mechanistic insight into how their expressions are controlled. Importantly, VGLL1 regulation of EGFR and CD44high/+CD24low/- population is mediated by the hippo-transducer TAZ which exerts its oncogenic roles by binding and activating TEAD transcription factors. A crucial finding is that TEAD-binding domain of TAZ is dispensable for its regulation of EGFR and CD44high/+CD24low/- cells. Instead, VGLL1 stabilization of cytoplasmic TAZ is essential for these functions. Also, we show that VGLL1/TAZ restricts the surface expression of CD24 which contributes to the increased number of CD44high/+CD24low/- cells. In addition, we observed that VGLL1 represses AXL expression and suppresses claudin-low phenotype, and that is caused by the VGLL1 mediated nuclear expulsion of TAZ. Therefore, EGFR and AXL seem to represent two different breast tumor subtypes, and their differential expressions is controlled by VGLL1.

IMP3 Stabilization of WNT5B mRNA Facilitates TAZ Activation in Breast Cancer.

Insulin-like growth factor-2 mRNA-binding protein 3 (IMP3) is an oncofetal protein associated with many aggressive cancers and implicated in the function of breast cancer stem cells (CSCs). The mechanisms involved, however, are poorly understood. We observed that IMP3 facilitates the activation of TAZ, a transcriptional co-activator of Hippo signaling that is necessary for the function of breast CSCs. The mechanism by which IMP3 activates TAZ involves both mRNA stability and transcriptional regulation. IMP3 stabilizes the mRNA of an alternative WNT ligand (WNT5B) indirectly by repressing miR145-5p, which targets WNT5B, resulting in TAZ activation by alternative WNT signaling. IMP3 also facilitates the transcription of SLUG, which is necessary for TAZ nuclear localization and activation, by a mechanism that is also mediated by WNT5B. These results demonstrate that TAZ can be regulated by an mRNA-binding protein and that this regulation involves the integration of Hippo and alternative WNT-signaling pathways.

Main-chain polyacetal conjugates with HIF-1 inhibitors: temperature-responsive, pH-degradable drug delivery vehicles.

Main-chain polymer-drug conjugates are prepared from polyacetals (PA) and three hydrophobic diol-based HIF-1 inhibitors. The new conjugates are temperature-responsive with lower critical solution temperature (LCST) behavior and are intrinsically pH-degradable. While soluble in plasma at room temperature, they lose solubility above a target temperature that can be adjusted to virtually any temperature of physicological interest, providing mechanisms for site-specific delivery by active thermal targeting or temperature-induced gelation. The reverse phase transition temperature can be precisely tuned by proper choice of four structural variables that characterize the amphiphilic diol and divinyl ether monomers used in the synthesis, or by adjusting the content of drug incorporated within the polymer. These main-chain PA-drug conjugates also allow for site-specific controlled release as they degrade in acidic microenvironments such as tumors. The degradation rates increase with decreasing pH, degradation products are neutral, and pristine drug is released, without any remnants of the conjugation chemistry.

Preventing Alkyne-Alkyne (i.e., Glaser) Coupling Associated with the ATRP Synthesis of Alkyne-Functional Polymers/Macromonomers and for Alkynes under Click (i.e., CuAAC) Reaction Conditions.

Alkyne-functional polymers synthesized by ATRP exhibit bimodal molecular weight distributions indicating the occurrence of some undesirable side reaction. By modeling the molecular weight distributions obtained under various reaction conditions, we show that the side reaction is alkyne-alkyne (i.e., Glaser) coupling. Glaser coupling accounts for as much as 20% of the polymer produced, significantly compromising the polymer functionality and undermining the success of subsequent click reactions in which they are used. Glaser coupling does not occur during ATRP but during postpolymerization workup upon first exposure to air. Two strategies are reported that effectively eliminate these coupling reactions without the need for a protecting group for the alkyne-functional initiator: (1) maintaining low temperature post-ATRP upon exposure to air followed by immediate removal of copper catalyst; (2) adding excess reducing agents post-ATRP which prevent the oxidation of Cu(I) catalyst required by the Glaser coupling mechanism. Post-ATRP Glaser coupling was also influenced by the ATRP synthesis ligand used. The order of ligand activity for catalyzing Glaser coupling was: linear bidentate > tridentate > tetradentate. We find that Glaser coupling is not problematic in ARGET-ATRP of alkyne-terminated polymers because a reducing agent is present during polymerization, however the molecular weight distribution is broadened compared to ATRP due to the presence of oxygen. Glaser coupling can also occur for alkynes held under CuAAC reaction conditions but again can be eliminated by adding appropriate reducing agents.

Enhanced Spacer Length between Mannose Mimicking Shikimoyl and Quinoyl Headgroups and Hydrophobic Region of Cationic Amphiphile Increases Efficiency of Dendritic Cell Based DNA Vaccination: A Structure-Activity Investigation.

In the field of dendritic cell based genetic immunization, previously we showed that liposomes of cationic amphiphiles containing mannose-mimicking shikimoyl headgroup are promising DNA vaccine carriers for dendritic cell (DC) transfection. The present structure-activity study reports on the influence of spacer length (between mannose-mimicking headgroups and quaternary nitrogen centers) in modulating the DC-transfection efficiencies. Further, we report on the anti-melanoma immune response inducing properties of the promising cationic amphiphiles in syngeneic C57BL/6J mice under prophylactic settings.

ERß regulation of NF-kB activation in prostate cancer is mediated by HIF-1.

We examined the regulation of NF-κB in prostate cancer by estrogen receptor ß (ERß) based on the inverse correlation between p65 and ERß expression that exists in prostate carcinomas and reports that ERß can inhibit NF-κB activation, although the mechanism is not known. We demonstrate that ERß functions as a gate-keeper for NF-κB p65 signaling by repressing its expression and nuclear translocation. ERß regulation of NF-κB signaling is mediated by HIF-1. Loss of ERß or hypoxia stabilizes HIF-1α, which we found to be a direct driver of IKKß transcription through a hypoxia response element present in the promoter of the IKKß gene. The increase of IKKß expression in ERß-ablated cells correlates with an increase in phospho-IκBα and concomitant p65 nuclear translocation. An inverse correlation between the expression of ERß and IKKß/p65 was also observed in the prostates of ERß knockout (BERKO) mice, Gleason grade 5 prostate tumors and analysis of prostate cancer databases. These findings provide a novel mechanism for how ERß prevents NF-κB activation and raise the exciting possibility that loss of ERß expression is linked to chronic inflammation in the prostate, which contributes to the development of high-grade prostate cancer.

Prostate tumorigenesis induced by PTEN deletion involves estrogen receptor ß repression.

The role of ERß in prostate cancer is unclear, although loss of ERß is associated with aggressive disease. Given that mice deficient in ERß do not develop prostate cancer, we hypothesized that ERß loss occurs as a consequence of tumorigenesis caused by other oncogenic mechanisms and that its loss is necessary for tumorigenesis. In support of this hypothesis, we found that ERß is targeted for repression in prostate cancer caused by PTEN deletion and that loss of ERß is important for tumor formation. ERß transcription is repressed by BMI-1, which is induced by PTEN deletion and important for prostate tumorigenesis. This finding provides a mechanism for how ERß expression is regulated in prostate cancer. Repression of ERß contributes to tumorigenesis because it enables HIF-1/VEGF signaling that sustains BMI-1 expression. These data reveal a positive feedback loop that is activated in response to PTEN loss and sustains BMI-1.

IMP3 protein promotes chemoresistance in breast cancer cells by regulating breast cancer resistance protein (ABCG2) expression.

IMP3, a member of a family of insulin-like growth factor II (IGF-II) mRNA-binding proteins (IMPs), is expressed preferentially in triple-negative breast cancers, which are resistant to many chemotherapeutics. However, the mechanisms by which it impacts breast cancer have not been elucidated. We hypothesized a role for IMP3 in chemoresistance based on these observations. Depletion of IMP3 expression in triple-negative breast cancer cells increased their sensitivity to doxorubicin and mitoxantrone significantly but not to taxol. Given that doxorubicin and mitoxantrone are effluxed by breast cancer resistance protein (BCRP), we assessed whether IMP3 regulates BCRP. The data obtained demonstrate that IMP3 binds to BCRP mRNA and regulates BCRP expression. These findings are significant because they provide insight into the mechanism by which IMP3 contributes to aggressive cancers, and they highlight the potential for targeting this mRNA-binding protein for the clinical management of cancer.

Nondestructive characterization of Li+ ion-doped multifunctional poly(vinylidene fluoride)-g-poly(dimethyl amino ethyl methacrylate) by impedance spectroscopy.

Poly(vinylidene fluoride) (PVDF)-graft-poly(dimethyl amino ethyl methacrylate) (PDMAEMA) (PD copolymer) is produced via atom transfer radical polymerization from PVDF solution in N-methyl-2-pyrrolidone. PD copolymer is doped with 1% and 5% (w/w) Li(+) ion to produce PDLi1 and PDLi5 samples, respectively. In PD copolymer, the crystalline structure of PVDF changes from α polymorph to a mixture of α and ß polymorph, and it transforms completely to piezoelectric ß polymorph on doping with 1% (w/w) Li(+) ion. The impedance behavior of PVDF changes on grafting, and that of the PD graft copolymer also changes with increasing Li(+) ion dopant concentration. In the Nyquist plots, PVDF exhibits a straight line character, and a curvature has appeared in the PD graft copolymer; on doping the latter with Li(+) ion (1% w/w), the curvature increases and a semicircle is completed on 5% Li(+) doping. Fitting the data from the Z-view program, the Ohmic resistance of PDLi1 is found to be 78 MΩ having capacitance with constant phase element (CPE) = 1.38 nF while for the PDLi5 sample the resistance decreases to16.1 MΩ with a small increase in CPE to 1.46 nF. The modulus plane plots for PDLi1 and PDLi5 samples also exhibit only one peak supporting the presence of only one equivalent resistance-capacitance circuit with constant phase element in both PDLi1 and PDLi5 samples. Both the impedance and modulus vs frequency plots of PDLi1 and PDLi5 samples exhibit a single Debye peak suggesting isotropic nature of the samples. For PVDF and PDMAEMA, ac-conductivity increases linearly with angular frequency, but in the case of PDLi1 and PDLi5 samples, it remains at first invariant in the frequency range 1-10(2) Hz, and above 10(2) Hz, an increase in conductivity with frequency occurs obeying the double power law. In the temperature variation of conductivity, PVDF exhibits its typical insulating nature, and in the PD graft copolymer, the conductivity decreases with increase of temperature (metallic-like behavior) due to gradual breaking of supramolecular interaction. The temperature variation of ac-conductivity of the Li(+)-doped PD graft copolymer suggests that both the ionic and supramolecular contributions of conductivity operate; the former increases and the latter decreases with rise in temperature showing a maximum. The temperature-dependent FTIR spectra of PDLi1 and PDLi5 samples support the gradual breaking of supramolecular interactions with increase of temperature.

The use of RGDGWK-lipopeptide to selectively deliver genes to mouse tumor vasculature and its complexation with p53 to inhibit tumor growth.

In vivo selection of phage display libraries have been exploited successfully in the past to isolate various high affinity conformationally strained cyclic peptide ligands (CX(5-7)C, peptides flanked by a cysteine residue on each side) for integrin receptors capable of selectively homing to tumor vasculatures. Previously, such phase display library studies have shown that integrin alpha 5 beta 1 binds with high affinity to cyclic peptides containing CRGDGWC motif. Herein we show that a lipopeptide with just the RGDGW motif (without the flanking cysteine groups) covalently attached to the lysine residue of a monolysinylated cationic amphiphile (RGDGWK-lipopeptide 1) delivers genes to cultured cells preferably via alpha 5 beta 1 integrins. Importantly, remarkable tumor growth inhibition was observed when the electrostatic complex of the RGDGWK-lipopeptide 1 and the anti-cancer p53 gene was intravenously administered in C57BL/6J mice bearing the aggressive B16F10 tumor. Immunohistochemical staining of mice tumor cryosections with vasculature markers combined with monitoring expression of the green fluorescence protein in the same tumor cryosections revealed that the RGDGWK-lipopeptide 1 targets genes to tumor vasculatures. The colocalization of the TUNEL (terminal deoxyuridine triphosphate nick-end labeling, a widely used marker of apoptosis) and VE-cadherin (markers of tumor endothelial cells) positive cells in tumor cryosections support the notion that the remarkable tumor growth inhibition property of the RGDGWK-lipopeptide 1:p53 complex is initiated through apoptosis of the tumor endothelial cells.

Cationic amphiphiles: promising carriers of genetic materials in gene therapy.

The clinical success of gene therapy critically depends on the use of efficient and safe gene delivery reagents. The present tutorial review is aimed at inspiring young researchers and students to take up the unsolved challenges in using cationic amphiphiles as safe gene transfer reagents. The review highlights important structure-activity studies in the field to date including the use of cationic amphiphiles for receptor specific targeted gene therapy and for delivery of siRNAs in the emerging field of RNA interference.