Single-cell transcriptomics reveals the intra-tumoral heterogeneity and SQSTM1/P62 and Wnt/ß-catenin mediated epithelial to mesenchymal transition and stemness of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is characterized by the complex tumor microenvironment (TME) consisting of an abundance of mesenchymal stem cells (MSCs), which is known to facilitate epithelial-to-mesenchymal transition (EMT). The development of single-cell genomics is a powerful method for defining the intricate genetic landscapes of malignancies. In this study, we have employed single-cell RNA sequencing (scRNA-seq) to dissect the intra-tumoral heterogeneity and analyze the single-cell transcriptomic landscape to detect rare consequential cell subpopulations of significance. The scRNA-seq analysis of TNBC and Normal patient derived samples revealed that EMT markers and transcription factors were most upregulated in MSC population. Further, exploration of gene expression analysis among TNBC and Normal patient-derived MSCs ascertained the role of SQSTM1/P62 and Wnt/ß-catenin in TNBC progression. Wnt/ß-catenin and Wnt/PCP signaling pathways are prominent contributors of EMT, stemness, and cancer stem cell (CSC) properties of TNBC. SQSTM1/P62 cooperates with the components of the Wnt/PCP signaling pathway and is critically involved at the interface of autophagy and EMT. Moreover, siRNA targeting SQSTM1/P62 and inhibitor of Wnt/ß-catenin (FH535) in conjunction was used to explore molecular modification of EMT and stemness markers. Although SQSTM1/P62 is not crucial for cell survival, cytotoxicity assay revealed synergistic interaction between the siRNA/inhibitor. Modulation of these important pathways helped in reduction of expression of genes and proteins contributing to CSC properties. Gene and protein expression analysis revealed the induction of EMT to MET. Moreover, co-treatment resulted in inactivation of non-canonical Wnt VANGL2-JNK signaling axis. The synergistic impact of inhibition of SQSTM1/P62 and Wnt/ß-catenin signaling facilitates the development of a potential therapeutic regimen for TNBC.

Transport Behavior of Commercial Anticancer Drug Protein-Bound Paclitaxel (Paclicad) in a Micron-Sized Channel.

Protein-bound paclitaxel has been developed clinically as one of the most successful chemotherapy drugs for the treatment of a wide variety of cancers. However, these medications, due to their nanoscale properties, may often induce capillary blocking while migrating through minute blood vessels. Considering the detrimental impact of this restriction, we investigated the transport of protein-bound paclitaxel, Paclicad, in a 7 µm microchannel mimicking the identical mechanical confinement of the blood capillaries. The drug was reported to migrate through a constricted microchannel without obstruction at a solution flow rate of 20-50 µL/h. The onset of an agglomeration site was observed at higher flow rates of 70-90 µL/h, while complete capillary obstruction was observed at 100 µL/h. The mobility of the particles was also calculated, and the results suggested that the presence of varying cross-sections affects the mobility of the drug particles. The trajectory of the particle migration was observed to be less tortuous at the higher flow rate, but the tortuous nature appeared to increase with the presence of agglomeration sites in the flow field. The experimental results were also compared with the computational model of the drug particle. The drug particle was modeled both as Newtonian and as an FENE-P viscoelastic drop. The drop interface tracking was done by the VOF method using the open source software Basilisk. The particle displacement was better estimated by both the FENE-P and Newtonian model at a flow rate of 30 µL/h, while deviation was observed at a flow rate of 50 µL/h. The FENE-P model was observed to show higher deformation than the Newtonian model at both flow rates. The experimental results provided better insight into the agglomeration tendency of Paclicad, migrating through a constricted microchannel at higher flow rates. The numerical model could be further employed to understand the more complex intravenous transport of drugs.

Copper(I)-Mediated Cascade Annulation via Dual C-H/C-H Activation: Access to Benzo[a]carbazolic AEEgens.

A Cu(I)-mediated cascade cyclization/annulation of unprotected o-alkynylanilines with maleimides in one pot is developed. The protocol offers sequential formation of one C-N and two C-C bonds to deliver fused benzo[a]carbazoles having free NH skeletons. The annulated products display fluorescence emission in the range of 485-502 nm with a large Stokes shift and fluorescence lifetime of ∼17 ns. The annulated 3aa displays AEE behavior in the ethanol/hexane system and possesses marigold-flower-like morphology at the aggregated state. Cell viability assays enumerate biocompatible AEEgens, while their high intracellular fluorescence depicts cell imaging applicability.

Deciphering therapeutic potential of PEGylated recombinant PTEN-silver nanoclusters ensemble on 3D spheroids.

The therapeutic application of recombinant proteins is limited due to their inherent structural complexity. Additionally, screening of therapeutic potential of protein products requires an appropriate testing platform to achieve biological relevance. Fabrication of three dimensional cultures bridges the gap between in vitro based monolayer cultures and clinical applications. In this perspective, glioblastoma U-87 MG and breast cancer MCF7 spheroids were generated to assess the therapeutic prospect of recombinant PTEN protein. PTEN bound to silver nanoclusters was encapsulated within PEG coating, which resulted in fabrication of spherical nanocarriers named as PTEN-nanocomposites. Internalization of PTEN-nanocomposites in the spheroids was confirmed by confocal microscopy. Upon uptake, PTEN-nanocomposites led to modulation of cyclins and apoptosis gene regulators culminating in cell cycle arrest and reduced cell viability as confirmed by calcein-AM/PI dual staining and alamar blue assay. Further, combination of tamoxifen and PTEN-nanocomposites on U-87 MG spheroids resulted in two-fold reduction of drug dosage. The study revealed that the monolayer culture results translated to the 3D culture as well, however higher dose of the recombinant PTEN was required for the spheroid system. The anti-proliferative role of PTEN-nanocomposites in a complex 3D environment augments its biological implication and paves the way for recombinant PTEN based therapeutic applications.

In silico screening and identification of potential drug against p300 acetyltransferase activity in breast cancer via drug repurposing approach.

Emerging evidence portray the involvement of epigenomic reprogramming in the onset and progression of several malignancies, including breast cancer. Histone acetyltransferase (HAT) p300 is a critical epigenetic regulator that acts as a transcription co-activator and regulates various cellular processes. p300 is overexpressed in breast cancer and promotes cellular invasion and survival, making it a promising druggable target. In this study, the relevance of p300 in different cancer pathways was established. Virtual screening of the FDA-approved drug library was carried out using molecular docking, and the top 10 potential repurposed drugs were identified. Further, recalculation of binding free energy of drug-p300 complexes was carried out using molecular mechanics Poisson-Boltzmann and surface area (MM-PBSA) method after molecular dynamic simulation. Based on molecular dynamic simulation parameters and binding free energy analysis, two drugs, namely Netarsudil (-305.068 kJ/mol) and Imatinib (-260.457 kJ/mol), were identified as potential repurposed drugs to inhibit the activity of p300. In conclusion, these findings suggest, Netarsudil and Imatinib might be a potential repurposed drug to combat breast cancer via p300 inhibition.Communicated by Ramaswamy H. Sarma.

Proline selective labeling via on-site construction of naphthoxazole (NapOx).

Chemoselective construction of naphthoxazoles (NapOx) via a three-component annulation reaction enables proline selective labeling of peptides in solution or in solid-phase synthesis. The fluorogenic peptides possess low cytotoxicity, efficient cell membrane permeability and excellent bioimaging potential for biomedical applications.

Tweaking EMT and MDR dynamics to constrain triple-negative breast cancer invasiveness by EGFR and Wnt/ß-catenin signaling regulation.

PURPOSE: Due to a lack of effective targeted therapies, patients with metastatic triple-negative breast cancer (TNBC) have poor clinical outcomes. Epithelial to mesenchymal transition (EMT) is known to contribute to cancer progression, invasiveness and multidrug resistance (MDR). There is a strong correlation between various drug efflux mechanisms, cancer stem cells and tumor microenvironments, which in turn is synchronized by complex signaling crosstalk between EMT and MDR. We hypothesize that combining these regulatory connections with targeted combinatorial therapies may be an effective approach to annihilate the progression/metastasis of TNBC. METHODS: AlamarBlue assays were used to depict TNBC cell viability, whereas flow cytometry was used to detect apoptotic cell populations, reactive-oxygen species (ROS) levels as well as mitochondrial depolarization. qRT-PCR, Western blotting and confocal microscopy were used to provide molecular-level information of the genes and proteins involved. RESULTS: Our initial analyses showed that targeting EGFR by either erlotinib (EGFR inhibitor) or lapatinib (EGFR/HER-2 inhibitor) alone was ineffective against TNBC. Interestingly, we subsequently found that a low dose of lapatinib did act as a substrate rather than as an inhibitor facilitating EMT and MDR, leading to metastasis. Additional gene expression studies indicated that co-targeting the EGFR and Wnt/ß-catenin pathways with lapatinib and XAV939 (a tankyrase inhibitor) promoted mesenchymal to epithelial transition (MET). Application of these inhibitors led to a 5.62-fold increase in the epithelial marker E-cadherin and a 3.33-fold decrease in the stemness marker EpCAM, with concomitant 1.5-fold and 3.22-fold reductions in the ABC transporters ABCB1 and ABCG2, respectively. These co-targeting effects resulted in overcoming EMT and MDR, which in turn was highlighted by reduced levels of pEGFR, pAKT, pMAPK, pSTAT-3, pGSK-3ß and ß-catenin. CONCLUSIONS: Our data indicate that the synergistic action of targeting both the EGFR and Wnt/ß-catenin signaling pathways in TNBC cells may open up new avenues for combatting this disease.

Transferrin Coated d-penicillamine-Au-Cu Nanocluster PLGA Nanocomposite Reverses Hypoxia-Induced EMT and MDR of Triple-Negative Breast Cancers.

Triple-negative breast cancer (TNBC), the most aggressive subtype of breast cancer, lacks effective targeted therapies due to negative expression of the targetable bioreceptors. Additionally, hypoxic condition in solid tumors contributes to the epithelial to mesenchymal transition (EMT), which aggravates cancer progression, multidrug resistance (MDR), migration, and stemness of the TNBC. A therapeutic module has been established in this regard by coating PLGA nanoparticle with d-penicillamine templated Au-Cu bimetallic nanoclusters. Further, the resultant nanomaterials were coated with recombinant transferrin protein to specifically target transferrin receptor overexpressing TNBC. The synthesized nanocomposites showed strong orange emission band at 630 nm with fluorescence quantum yield of 2%, rendering it suitable for theranostic applications. Experimental results demonstrated efficient cellular internalization and significant innate anti-cell proliferative potential of the nanocomposites. The fabricated nanocomposites were also able to induce cell death in spheroids, which was confirmed by live/dead dual staining results. Furthermore, when EMT-induced TNBC cells were treated with nanocomposites, they generated reactive oxygen species (ROS), depolarized the mitochondrial membrane potential, and induced apoptosis. Gene expression by real-time PCR indicated that treatment of EMT-induced TNBC cells with nanocomposites facilitated mesenchymal to epithelial transition (MET). In MDA-MB-468 cells, treatment with nanocomposites resulted in a 1.35-fold rise in E-cadherin an epithelial marker and a 1.36-fold decrease in vimentin a mesenchymal marker. Similarly, 2.87-fold and 1.76-fold decrease in stemness markers ALDH1A3 and EpCAM were observed in MDA-MB-231. Furthermore, 4.63-fold decrease in expression of ABCC1, a prominent contributor of MDR, was observed in MDA-MB-231. Protein expression studies revealed that nanocomposites reduced p-STAT-3 by 1.61-fold in MDA-MB-231 and by 7.8-fold in MDA-MB-468. Importantly, nanocomposites downregulated the expression of ß-catenin by 3-fold in MDA-MB-231 and by 3.11-fold in MDA-MB-468. Downregulation of EMT with concomitant alteration of STAT-3 and ß-catenin signaling pathways led to reduced migration ability of the TNBC cells.

Studying in vitro phagocytosis of apoptotic cancer cells by recombinant GMCSF-treated RAW 264.7 macrophages.

Granulocyte macrophage colony stimulating factor (GMCSF), a therapeutically important cytokine that helps in the proliferation of macrophages, was recombinantly expressed in E. coli BL21 and purified as a GST-tagged protein. Cell viability assay demonstrated significant enhancement in proliferation of RAW 264.7 (murine macrophage) in presence of GMCSF. In vitro activation of macrophages was carried out by lipopolysaccharide (LPS) or pyrogallol and probed by the generation of reactive oxygen species (ROS). Following the induction of apoptosis in A549 lung cancer cells with anticancer drug cisplatin (at 25µM), apoptotic cancer cells were effectively phagocytosed by the recombinant GMCSF-treated and exogenously activated RAW 264.7 cells as observed in fluorescence microscopic images. The current findings attribute possible role of GMCSF as adjuvant in scavenging treated cancer cells.