Lipidome-based Targeting of STAT3-driven Breast Cancer Cells Using Poly-l-glutamic Acid-coated Layer-by-Layer Nanoparticles.

The oncogenic transcription factor STAT3 is aberrantly activated in 70% of breast cancers, including nearly all triple-negative breast cancers (TNBCs). Because STAT3 is difficult to target directly, we considered whether metabolic changes driven by activated STAT3 could provide a therapeutic opportunity. We found that STAT3 prominently modulated several lipid classes, with most profound effects on N-acyl taurine and arachidonic acid, both of which are involved in plasma membrane remodeling. To exploit these metabolic changes therapeutically, we screened a library of layer-by-layer (LbL) nanoparticles (NPs) differing in the surface layer that modulates interactivity with the cell membrane. We found that poly-l-glutamic acid (PLE)-coated NPs bind to STAT3-transformed breast cancer cells with 50% greater efficiency than to nontransformed cells, and the heightened PLE-NP binding to TNBC cells was attenuated by STAT3 inhibition. This effect was also observed in densely packed three-dimensional breast cancer organoids. As STAT3-transformed cells show greater resistance to cytotoxic agents, we evaluated whether enhanced targeted delivery via PLE-NPs would provide a therapeutic advantage. We found that cisplatin-loaded PLE-NPs induced apoptosis of STAT3-driven cells at lower doses compared with both unencapsulated cisplatin and cisplatin-loaded nontargeted NPs. In addition, because radiation is commonly used in breast cancer treatment, and may alter cellular lipid distribution, we analyzed its effect on PLE-NP-cell binding. Irradiation of cells enhanced the STAT3-targeting properties of PLE-NPs in a dose-dependent manner, suggesting potential synergies between these therapeutic modalities. These findings suggest that cellular lipid changes driven by activated STAT3 may be exploited therapeutically using unique LbL NPs.

GSE4-loaded nanoparticles a potential therapy for lung fibrosis that enhances pneumocyte growth, reduces apoptosis and DNA damage.

Idiopathic pulmonary fibrosis is a lethal lung fibrotic disease, associated with aging with a mean survival of 2-5 years and no curative treatment. The GSE4 peptide is able to rescue cells from senescence, DNA and oxidative damage, inflammation, and induces telomerase activity. Here, we investigated the protective effect of GSE4 expression in vitro in rat alveolar epithelial cells (AECs), and in vivo in a bleomycin model of lung fibrosis. Bleomycin-injured rat AECs, expressing GSE4 or treated with GSE4-PLGA/PEI nanoparticles showed an increase of telomerase activity, decreased DNA damage, and decreased expression of IL6 and cleaved-caspase 3. In addition, these cells showed an inhibition in expression of fibrotic markers induced by TGF-ß such as collagen-I and III among others. Furthermore, treatment with GSE4-PLGA/PEI nanoparticles in a rat model of bleomycin-induced fibrosis, increased telomerase activity and decreased DNA damage in proSP-C cells. Both in preventive and therapeutic protocols GSE4-PLGA/PEI nanoparticles prevented and attenuated lung damage monitored by SPECT-CT and inhibited collagen deposition. Lungs of rats treated with bleomycin and GSE4-PLGA/PEI nanoparticles showed reduced expression of α-SMA and pro-inflammatory cytokines, increased number of pro-SPC-multicellular structures and increased DNA synthesis in proSP-C cells, indicating therapeutic efficacy of GSE4-nanoparticles in experimental lung fibrosis and a possible curative treatment for lung fibrotic patients.

CDK5RAP3 is a co-factor for the oncogenic transcription factor STAT3.

The transcription factor STAT3 regulates genes governing critical cellular processes such as proliferation, survival, and self-renewal. While STAT3 transcriptional function is activated rapidly and transiently in response to physiologic signals, through a variety of mechanisms it can become constitutively activated in the pathogenesis of cancer. This leads to chronic expression of genes that underlie malignant cellular behavior. However, STAT3 is known to interact with other proteins, which may modulate its function. Understanding these interactions can provide insights into novel aspects of STAT3 function and may also suggest strategies to therapeutically target the large number of cancers driven by constitutively activated STAT3. To identify critical modulators of STAT3 transcriptional function, we performed an RNA-interference based screen in a cell-based system that allows quantitative measurement of STAT3 activity. From this approach, we identified CDK5 kinase regulatory-subunit associated protein 3 (CDK5RAP3) as an enhancer of STAT3-dependent gene expression. We found that STAT3 transcriptional function is modulated by CDK5RAP3 in cancer cells, and silencing CDK5RAP3 reduces STAT3-mediated tumorigenic phenotypes including clonogenesis and migration. Mechanistically, CDK5RAP3 binds to STAT3-regulated genomic loci, in a STAT3-dependent manner. In primary human breast cancers, the expression of CDK5RAP3 expression was associated with STAT3 gene expression signatures as well as the expression of individual STAT3 target genes. These findings reveal a novel aspect of STAT3 transcriptional function and potentially provide both a biomarker of enhanced STAT3-dependent gene expression as well as a unique mechanism to therapeutically target STAT3.

GSE4 peptide suppresses oxidative and telomere deficiencies in ataxia telangiectasia patient cells.

Ataxia telangiectasia (AT) is a genetic disease caused by mutations in the ATM gene but the mechanisms underlying AT are not completely understood. Key functions of the ATM protein are to sense and regulate cellular redox status and to transduce DNA double-strand break signals to downstream effectors. ATM-deficient cells show increased ROS accumulation, activation of p38 protein kinase, and increased levels of DNA damage. GSE24.2 peptide and a short derivative GSE4 peptide corresponding to an internal domain of Dyskerin have proved to induce telomerase activity, decrease oxidative stress, and protect from DNA damage in dyskeratosis congenita (DC) cells. We have found that expression of GSE24.2 and GSE4 in human AT fibroblast is able to decrease DNA damage, detected by γ-H2A.X and 53BP1 foci. However, GSE24.2/GSE4 expression does not improve double-strand break signaling and repair caused by the lack of ATM activity. In contrast, they cause a decrease in 8-oxoguanine and OGG1-derived lesions, particularly at telomeres and mitochondrial DNA, as well as in reactive oxygen species, in parallel with increased expression of SOD1. These cells also showed lower levels of IL6 and decreased p38 phosphorylation, decreased senescence and increased ability to divide for longer times. Additionally, these cells are more resistant to treatment with H202 and the radiomimetic-drug bleomycin. Finally, we found shorter telomere length (TL) in AT cells, lower levels of TERT expression, and telomerase activity that were also partially reverted by GSE4. These observations suggest that GSE4 may be considered as a new therapy for the treatment of AT that counteracts the cellular effects of high ROS levels generated in AT cells and in addition increases telomerase activity contributing to increased cell proliferation.

The STAT3 Target Gene TNFRSF1A Modulates the NF-κB Pathway in Breast Cancer Cells.

The transcription factor STAT3 is activated inappropriately in 70% of breast cancers, most commonly in triple negative breast cancer (TNBC). Although the transcriptional function of STAT3 is essential for tumorigenesis, the key target genes regulated by STAT3 in driving tumor pathogenesis have remained unclear. To identify critical STAT3 target genes, we treated TNBC cell lines with two different compounds that block STAT3 transcriptional function, pyrimethamine and PMPTP. We then performed gene expression analysis to identify genes whose expression is strongly down-regulated by both STAT3 inhibitors. Foremost among the down-regulated genes was TNFRSF1A, which encodes a transmembrane receptor for TNFα. We showed that STAT3 binds directly to a regulatory region within the TNFRSF1A gene, and that TNFRSF1A levels are dependent on STAT3 function in both constitutive and cytokine-induced models of STAT3 activation. Furthermore, TNFRSF1A is a major mediator of both basal and TNFα-induced NF-κB activity in breast cancer cells. We extended these findings to primary human breast cancers, in which we found that high TNFRSF1A transcript levels correlated with STAT3 activation. In addition, and consistent with a causal role, increased TNFRSF1A expression was associated with an NF-κB gene expression in signature in breast cancers. Thus, TNFRSF1A is a STAT3 target gene that regulates the NF-κB pathway. These findings reveal a novel functional crosstalk between STAT3 and NF-κB signaling in breast cancer. Furthermore, elevated TNFRSF1A levels may predict a subset of breast tumors that are sensitive to STAT3 transcriptional inhibitors, and may be a biomarker for response to inhibition of this pathway.

Optoacoustic imaging enabled biodistribution study of cationic polymeric biodegradable nanoparticles.

Nanosized contrast agents for molecular imaging have attracted widespread interest for diagnostic applications with high resolution in medicine. However, many solid nanoparticles exhibit a great potential to induce toxicity, hindering their use for clinical applications. On the other hand, near-infrared (NIR) dyes have also been used for extensive biological applications, but show some limitations due to their poor aqueous stability, tendency to aggregation and rapid elimination from the body. An alternative proposed in this work to overcome these limitations is the use of NIR dye-loaded nanoparticles. Here we introduce nanoparticles constructed with poly(D,L-lactide-co-glycolic acid) (PLGA), a biodegradable and biocompatible polymer widely used for biomedical applications, attached to the polycation polyethyleneimine (PEI) to obtain positively charged nanoparticles. The in vivo biodistribution of the cationic PEI-PLGA nanoparticles was investigated after administration through three different routes (intravenous, intraperitoneal and subcutaneous) using multispectral optoacoustic tomography (MSOT). The prepared nanoparticles exhibited good colloidal stability and adequate optical properties for optoacoustic imaging. The in vivo biodistribution assays indicated a strong accumulation of the particles in the liver and spleen, and retention in these organs for at least 24 h. Therefore, these nanoparticles could find promising applications in MSOT due to a sharp and characteristic optoacoustic spectrum and high optoacoustic signal generation, and become a promising building block for theranostic strategies.

Development of surface modified biodegradable polymeric nanoparticles to deliver GSE24.2 peptide to cells: a promising approach for the treatment of defective telomerase disorders.

The aim of the present study was to develop a novel strategy to deliver intracellularly the peptide GSE24.2 for the treatment of Dyskeratosis congenita (DC) and other defective telomerase disorders. For this purpose, biodegradable polymeric nanoparticles using poly(lactic-co-glycolic acid) (PLGA NPs) or poly(lactic-co-glycolic acid)-poly ethylene glycol (PLGA-PEG NPs) attached to either polycations or cell-penetrating peptides (CPPs) were prepared in order to increase their cellular uptake. The particles exhibited an adequate size and zeta potential, with good peptide loading and a biphasic pattern obtained in the in vitro release assay, showing an initial burst release and a later sustained release. GSE24.2 structural integrity after encapsulation was assessed using SDS-PAGE, revealing an unaltered peptide after the NPs elaboration. According to the cytotoxicity results, cell viability was not affected by uncoated polymeric NPs, but the incorporation of surface modifiers slightly decreased the viability of cells. The intracellular uptake exhibited a remarkable improvement of the internalization, when the NPs were conjugated to the CPPs. Finally, the bioactivity, addressed by measuring DNA damage rescue and telomerase reactivation, showed that some formulations had the lowest cytotoxicity and highest biological activity. These results proved that GSE24.2-loaded NPs could be delivered to cells, and therefore, become an effective approach for the treatment of DC and other defective telomerase syndromes.

Development and validation of a rapid HPLC method for the quantification of GSE4 peptide in biodegradable PEI-PLGA nanoparticles.

In this work a high performance liquid chromatographic (HPLC) method has been developed and validated for the content determination of GSE4 peptide in PEI-PLGA nanoparticles. Chromatographic separation was performed on a C18 column, and a gradient elution with a mobile phase composed of methanol and 0.1% aqueous trifluoroacetic acid (TFA) solution, at a flow rate of 1ml/min, was used. GSE4 peptide identification was made by fluorescence detection at 290nm. The elution of methanol:TFA was initially maintained at (20:80, v/v) for one min and the gradient changed to (80:20, v/v) in 6min. This ratio was then followed by isocratic elution at (80:20, v/v) during another min and for further 3min it was linearly modified to (20:80, v/v). The developed method was validated according to the ICH guidelines, being specific, linear in the range 10-100µg/ml (R(2)=0.9996), precise, exhibiting good inter-day and intra-day precision reflected by the relative standard deviation values (less than 3.88%), accurate, with a recovery rate of 100.18±0.95%, and stable for 48h at 5°C or at RT when encapsulated in nanoparticles. The method was simple, fast, and successfully used to determine the peptide content in GSE4-loaded PEI-PLGA nanoparticles.