Nuclear actin (N-actin) is known to participate in the regulation of gene expression. We showed previously that N-actin levels mediate the growth and quiescence of mouse epithelial cells in response ...to laminin-111 (LN1), a component of the mammary basement membrane (BM). We know that BM is defective in malignant cells, and we show here that it is the LN1/N-actin pathway that is aberrant in human breast cancer cells, leading to continuous growth. Photobleaching assays revealed that N-actin exit in nonmalignant cells begins as early as 30 min after LN1 treatment. LN1 attenuates the PI3K pathway leading to upregulation of exportin-6 (XPO6) activity and shuttles actin out of the nucleus. Silencing XPO6 prevents quiescence. Malignant cells are impervious to LN1 signaling. These results shed light on the crucial role of LN1 in quiescence and differentiation and how defects in the LN1/PI3K/XPO6/N-actin axis explain the loss of tissue homeostasis and growth control that contributes to malignant progression.
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•N-actin levels control transcription and proliferation of normal mammary cells•Laminin-111 (LN1) induces a rapid decrease of nuclear actin mediated by XPO6•LN1 attenuates PI3K, leading to upregulation of XPO6 activity•The LN1/N-actin pathway is aberrant in malignant cells, causing uncontrolled growth
Fiore et al. show that laminin-111 (LN1) induces a drastic decrease of nuclear actin in human mammary epithelial cells in a process mediated by XPO6 and required for acquisition of cellular quiescence. The LN1/XPO6/N-actin pathway is abnormal in malignant cells that are unresponsive to LN1 and proliferate uncontrollably.
Some anticancer treatments may cause Multidrug Resistance (MDR). In these cases, cells pump the drug out of the intracellular environment, thereby preventing drug effects. Several strategies have ...been used to avoid MDR, including using two or more drugs at low concentrations to increase the sensitivity of cells to treatment. We present an effective, cheap, fast microfluidic alternative to test two drugs simultaneously using a reversible sealing and reusable device to determine the optimal concentration. We used the rugs doxorubicin (DOX) and paclitaxel (PXT) as proof of concept. The microdevice allows the testing of two drugs in real time. Furthermore, running two experiments in sextuplicates and control in the same microchip is possible. We used two combinations of drugs, varying the drug concentration (C
1
= 0.010 mg.mL
− 1
DOX and 0.002.mL
− 1
mg PXT, C
2
= 0.010 mg.mL
− 1
DOX and 0.004 mg.mL
− 1
PXT), and evaluated cell death over time. The intermediate drug concentrations were more efficient, reducing the time required to decrease the viability of breast tumor cells, MCF-7 (C
1
= 180 and C
2
= 120). In further analysis, the microdevice also allowed characterization of the effects of the drugs (antagonist, synergic, or additive). This microdevice is a reliable tool for estimating the different combinations of two drug concentrations in a single assay simply and quickly.
Surface attachment of a planktonic bacteria, mediated by adhesins and extracellular polymeric substances (EPS), is a crucial step for biofilm formation. Some pathogens can modulate cell adhesiveness, ...impacting host colonization and virulence. A framework able to quantify cell-surface interaction forces and their dependence on chemical surface composition may unveil adhesiveness control mechanisms as new targets for intervention and disease control. Here we employed InP nanowire arrays to dissect factors involved in the early stage biofilm formation of the phytopathogen Xylella fastidiosa. Ex vivo experiments demonstrate single-cell adhesion forces up to 45 nN, depending on the cell orientation with respect to the surface. Larger adhesion forces occur at the cell poles; secreted EPS layers and filaments provide additional mechanical support. Significant adhesion force enhancements were observed for single cells anchoring a biofilm and particularly on XadA1 adhesin-coated surfaces, evidencing molecular mechanisms developed by bacterial pathogens to create a stronger holdfast to specific host tissues.
Several studies reported the concerted and mutual communication between the prostate epithelium and stroma, which determines the final organ architecture and function, but gets awry in cancer. ...Deciphering the mechanisms involved in this communication is crucial to find new therapeutic strategies. HS sequesters a number of secreted growth factors and cytokines, controlling their bioavailability to the target cells, suggesting that HS is an important regulator of the extracellular matrix (ECM) and a key player in the cell‐cell and cell‐microenvironment communication during prostate morphogenesis and physiology. We propose that by controlling HS biosynthesis and sulfation pattern, as well as the cleavage of the HS chain and/or the shedding of proteoglycans, epithelial and stromal cells are able to precisely tune the availability of signaling molecules and modulate ligand‐receptor interaction and intracellular signal transduction.
Key Findings
Embryonic and early postnatal prostate development is summarized
Heparan sulfate ability to concentrate signaling molecules is put in the context of prostate development
A model is proposed to include the participation of both epithelial and stromal cells in the synthesis and remodeling of the extracellular matrix and, hence, the content and distribution of heparan sulfate
Localized release of nucleic acid therapeutics is essential for many biomedical applications, including gene therapy, tissue engineering, and medical implant coatings. We applied the ...substrate-mediated transfection and layer-by-layer (LbL) technique to achieve an efficient local gene delivery. In the experiments presented herein, we embeded lipoplexes containing plasmid DNA encoding for enhanced green fluorescent protein (pEGFP) within polyelectrolyte alginate-based microgels composed of poly(allylamine hydrochloride) (PAH), chondroitin sulfate (CS), and poly-l-lysine (PLL) with diameters between 70 and 90 μm. Droplet-based microfluidics was used as the main process to produce the alginate (ALG)-based microgels with discrete size, shape, and low coefficient of variation. The physicochemical and morphological properties of the polyelectrolyte microgels were characterized via optical microscopy, scanning electron microscopy (SEM), and zeta potential analysis. We found that polyelectrolyte microgels provide low cytotoxicity and cell-material interactions (adhesion, spreading, and proliferation). In addition, the microsystem showed the ability to load lipoplexes and a loading efficiency equal to 83%, and it enabled in vitro surface-based transfection of MCF-7 cells. This approach provides a new suitable route for cell adhesion and local gene delivery.
Prostate cancer (PCa) is the leading cause of cancer-associated mortality in men, and new biomarkers are still needed. The expression pattern and protein tissue localization of proteoglycans of the ...syndecan family (SDC 1–4) and syntenin-1 (SDCBP) were determined in normal and prostatic tumor tissue from two genetically engineered mouse models and human prostate tumors. Studies were validated using SDC 1–4 and SDCBP mRNA levels and patient survival data from The Cancer Genome Atlas and CamCAP databases. RNAseq showed increased expression of Sdc1 in Pb-Cre4/Ptenf/f mouse Pca and upregulation of Sdc3 expression and downregulation of Sdc2 and Sdc4 when compared to the normal prostatic tissue in Pb-Cre4/Trp53f/f-;Rb1f/f mouse tumors. These changes were confirmed by immunohistochemistry. In human PCa, SDC 1–4 and SDCBP immunostaining showed variable localization. Furthermore, Kaplan–Meier analysis showed that patients expressing SDC3 had shorter prostate-specific survival than those without SDC3 expression (log-rank test, p = 0.0047). Analysis of the MSKCC-derived expression showed that SDC1 and SDC3 overexpression is predictive of decreased biochemical recurrence-free survival (p = 0.0099 and p = 0.045, respectively), and SDC4 overexpression is predictive of increased biochemical recurrence-free survival (p = 0.035). SDC4 overexpression was associated with a better prognosis, while SDC1 and SDC3 were associated with more aggressive tumors and a worse prognosis.
Prostate cancer (PCa) is the second most common cause of mortality among men. Tumor secretome is a promising strategy for understanding the biology of tumor cells and providing markers for disease ...progression and patient outcomes. Here, transcriptomic-based secretome analysis was performed on the PCa tumor transcriptome of Genetically Engineered Mouse Model (GEMM) Pb-Cre4/Ptenf/f mice to identify potentially secreted and membrane proteins—PSPs and PMPs. We combined a selection of transcripts from the GSE 94574 dataset and a list of protein-coding genes of the secretome and membrane proteome datasets using the Human Protein Atlas Secretome. Notably, nine deregulated PMPs and PSPs were identified in PCa (DMPK, PLN, KCNQ5, KCNQ4, MYOC, WIF1, BMP7, F3, and MUC1). We verified the gene expression patterns of Differentially Expressed Genes (DEGs) in normal and tumoral human samples using the GEPIA tool. DMPK, KCNQ4, and WIF1 targets were downregulated in PCa samples and in the GSE dataset. A significant association between shorter survival and KCNQ4, PLN, WIF1, and F3 expression was detected in the MSKCC dataset. We further identified six validated miRNAs (mmu-miR-6962-3p, mmu-miR- 6989-3p, mmu-miR-6998-3p, mmu-miR-5627-5p, mmu-miR-15a-3p, and mmu-miR-6922-3p) interactions that target MYOC, KCNQ5, MUC1, and F3. We have characterized the PCa secretome and membrane proteome and have spotted new dysregulated target candidates in PCa.
The WFDC1 gene is frequently down-regulated or lost in prostate cancer, and the encoded protein, ps20, has been implicated in epithelial cell behaviour and angiogenesis. However, ps20 remains largely ...uncharacterised with respect to its structure and interacting partners. This study characterised the evolution, functionality and structural characteristics of WFDC1/ps20 using phylogenetic reconstruction and other computational approaches. Bayesian phylogenetic analyses suggested that ps20 appeared in a common ancestor of deuterostomes-protostomes. The rate of evolutionary change within the coding regions of vertebrate WFDC1 genes and the synteny conservation in mammals differed from that of other vertebrate clades, indicating a possible functional diversity of ps20 homologues. A gene set enrichment analysis of the genes around WFDC1 (conserved synteny) showed functional relationships between the WFDC1, CDH13, CRISPLD2, IRF8 and TFPI2 genes. The molecular evolution of ps20 has been driven by purifying selection, particularly in the segments corresponding to exons 3 and 4, which encode the most conserved regions of the protein. A co-evolution analysis showed that residues within these regions co-vary with each other during the evolution of ps20. These results show that the regions corresponding to exons 3 and 4 are ps20-specific structure-function modules. Homology modelling of the exon 2-encoded polypeptide and subsequent dynamics calculus using a Gaussian network model showed that residues with high conformational flexibility are part of a loop region involved in protein-protein recognition, given the similarity with other serine protease inhibitors. Residues C96, R94, L105, and C66 are critical for the integrity and functionality of this ps20 region.
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•The small protein ps20 is coded by the WFDC1 gene, which originated in a deuterostomes-protostomes common ancestor.•Evolutionary rate in mammals differed from other clades, due to its function in reproduction (i.e. expression by both prostate and placenta).•Synteny analysis revealed functional relationships among the WFDC1, CDH13, CRISOLD2, IRF8 and PFPI2 genes.•Conservation rates varied, suggesting that exons 3 and 4-coded peptides are WFDC1/ps20-specific functional modules.
This study evaluated the association of mucinous metaplasia (MM) with tumor cell proliferation, androgen receptor (AR) expression and invasiveness in Pten conditional knockout mice and the prognostic ...value of MM markers for patients with PCa.
Prostatic lobes samples from genetic engineered mouse model Ptenf/f and Pb-Cre4/Ptenf/f were submitted for histopathological analysis and tissue expression of AR, the proliferation marker Ki67, alpha-smooth muscle actin, and laminin. RNAseq data of prostatic lobes samples were analyzed searching for MM gene expression patterns. We also investigated gene and protein expression related to MM in human PCa public databases.
All knockout animals analyzed showed at least one area of stroma-invading MM, which was absent in the control animals. The tumoral regions of MM showed a proliferative index 5 times higher than other tumoral areas and low expression of the AR (less than 20% of the cells were AR-positive). Disrupted basement membrane areas were observed in MM. The mouse and human PCa transcriptomes exhibited increased expression of the MM markers such as MUC1, MUC19, MUC4, MUC5AC, MUC5B, and TFF3. Gene expression profile was associated with castration-resistant prostate cancer (CRPC) and with a lower probability of freedom from biochemical recurrence.
The expression of goblet cell genes, such as MUC1, MUC5AC, MUC5B, and TFF3 have significant prognostic value for PCa patients and represent another class of potential therapeutic targets.
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•Mucinous metaplasia is highly frequent in Pten conditional knockout mouse model for prostate cancer.•Mucinous metaplasia presents cells with high proliferation index and without androgen receptor expression.•Mucinous metaplasia can be source of undifferentiated androgen-insensitive cells.•Mucin-family genes expression levels have prognostic value for patients with prostate cancer.
Wild-type or engineered bacteriophages have been reported as therapeutic agents in the treatment of several types of diseases, including cancer. They might be used either as naked phages or as ...carriers of antitumor molecules. Here, we evaluate the role of bacteriophages M13 and T4 in modulating the expression of genes related to cell adhesion, growth, and survival in the androgen-responsive LNCaP prostatic adenocarcinoma-derived epithelial cell line. LNCaP cells were exposed to either bacteriophage M13 or T4 at a concentration of 1 × 10
pfu/mL, 1 × 10
pfu/mL, and 1 × 10
pfu/mL for 24, 48, and 72 h. After exposure, cells were processed for general morphology, cell viability assay, and gene expression analyses. Neither M13 nor T4 exposure altered cellular morphology, but both decreased the MTT reduction capacity of LNCaP cells at different times of treatment. In addition, genes
,
,
,
,
,
, and
were significantly up-regulated, whilst the genes
,
,
, and
were down-regulated. Our results show that bacteriophage M13 and T4 interact with LNCaP cells and effectively promote gene expression changes related to anchorage-dependent survival and androgen signaling. In conclusion, phage therapy may increase the response of PCa treatment with
pathway inhibitors.
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