Structure-based design of an urokinase-type plasminogen activator receptor–derived peptide inhibiting cell migration and lung metastasis

The urokinase-type plasminogen activator receptor (uPAR) plays a central role in sustaining the malignant phenotype and promoting tumor metastasis. The Ser88-Arg-Ser-Arg-Tyr92 is the minimum chemotactic sequence of uPAR required to induce the same intracellular signaling as its ligand uPA. Here, we describe the generation of new peptide inhibitors of cell migration and invasion derived from SRSRY by a drug design approach. Ac-Arg-Glu-Arg-Phe-NH2 (i.e., RERF), which adopts a turned structure in solution, was selected for its ability to potently prevent SRSRY-directed cell migration. Fluorescein-RERF associates with very high affinity to RBL-2H3 rat basophilic leukemia cells expressing the human formyl peptide receptor (FPR). Accordingly, femtomolar concentrations of RERF prevent agonist-dependent internalization of FPR and inhibit N-formyl-Met-Leu-Phe–dependent migration in a dose-dependent manner. In the absence of FPR, fluorescein-RERF binds to cell surface at picomolar concentrations in an αv integrin–dependent manner. The involvement of vitronectin receptor is further supported by the findings that 100 pmol/L RERF selectively inhibits vitronectin-dependent RBL-2H3 cell migration and prevents SRSRY-triggered uPAR/αv association. Furthermore, RERF reduces the speed of wound closure and the extent of Matrigel invasion by human fibrosarcoma HT1080 cells without affecting cell proliferation. Finally, a 3- to 5-fold reduction of lung metastasis number and size in nude mice following i.v. injection of green fluorescent protein–expressing HT1080 cells in the presence of 3.32 mg/kg RERF is observed. Our findings indicate that RERF effectively prevents malignant cell invasion in vivo with no signs of toxicity and may represent a promising prototype drug for anticancer therapy

Modulation of Cellular Migration and Survival by c-Myc through the Downregulation of Urokinase (uPA) and uPA Receptor▿ †

It has been proposed that c-Myc proapoptotic activity accounts for most of its restraint of tumor formation. We established a telomerase-immortalized human epithelial cell line expressing an activatable c-Myc protein. We found that c-Myc activation induces, in addition to increased sensitivity to apoptosis, reductions in cell motility and invasiveness. Transcriptome analysis revealed that urokinase (uPA) and uPA receptor (uPAR) were strongly downregulated by c-Myc. Evidence is provided that the repression of uPA and uPAR may account for most of the antimigratory and proapoptotic activities of c-Myc. c-Myc is known to cooperate with Ras in cellular transformation. We therefore investigated if this cooperation could converge in the control of uPA/uPAR expression. We found that Ras is able to block the effects of c-Myc activation on apoptosis and cellular motility but not on cell invasiveness. Accordingly, the activation of c-Myc in the context of Ras expression had only minor influence on uPAR expression but still had a profound repressive effect on uPA expression. Thus, the differential regulation of uPA and uPAR by c-Myc and Ras correlates with the effects of these two oncoproteins on cell motility, invasiveness, and survival. In conclusion, we have discovered a novel link between c-Myc and uPA/uPAR. We propose that reductions of cell motility and invasiveness could contribute to the inhibition of tumorigenesis by c-Myc and that the regulation of uPA and uPAR expression may be a component of the ability of c-Myc to reduce motility and invasiveness

Protein Kinase A Activation Promotes Cancer Cell Resistance to Glucose Starvation and Anoikis.

Cancer cells often rely on glycolysis to obtain energy and support anabolic growth. Several studies showed that glycolytic cells are susceptible to cell death when subjected to low glucose availability or to lack of glucose. However, some cancer cells, including glycolytic ones, can efficiently acquire higher tolerance to glucose depletion, leading to their survival and aggressiveness. Although increased resistance to glucose starvation has been shown to be a consequence of signaling pathways and compensatory metabolic routes activation, the full repertoire of the underlying molecular alterations remain elusive. Using omics and computational analyses, we found that cyclic adenosine monophosphate-Protein Kinase A (cAMP-PKA) axis activation is fundamental for cancer cell resistance to glucose starvation and anoikis. Notably, here we show that such a PKA-dependent survival is mediated by parallel activation of autophagy and glutamine utilization that in concert concur to attenuate the endoplasmic reticulum (ER) stress and to sustain cell anabolism. Indeed, the inhibition of PKA-mediated autophagy or glutamine metabolism increased the level of cell death, suggesting that the induction of autophagy and metabolic rewiring by PKA is important for cancer cellular survival under glucose starvation. Importantly, both processes actively participate to cancer cell survival mediated by suspension-activated PKA as well. In addition we identify also a PKA/Src mechanism capable to protect cancer cells from anoikis. Our results reveal for the first time the role of the versatile PKA in cancer cells survival under chronic glucose starvation and anoikis and may be a novel potential target for cancer treatment

FSK-mediated attenuation of UPR is accompanied by an increase of membrane protein glycosylation.

<p>(<b>A-B)</b> UPR-related transcriptional data from microarray (A) and proteomic (B) data. (<b>C-G)</b> All the analyses are referred to Transformed cells and were performed at 72h (C-E) or 96h (F-G) of culture. (<b>C)</b> qPCR analysis of UPR mRNA levels in FSK-treated cells with respect to untreated cells. (<b>D)</b> Western blot analysis of UPR-related proteins. (<b>E)</b> Schematic representation of the HBP in which the expression levels of some HBP-related mRNAs have been indicated. The data are the ratio TF/T and are represented in color code (red up-regulated; yellow unchanged; green down-regulated). (<b>F)</b> FACS analysis of live cells stained with fluorochrome-conjugated ConcanavalinA. (<b>G)</b> Western blot analysis using a specific anti O-GlcNAc antibody and densitometric analysis of the film. All data represent the average of different experiments (n>3).</p

FSK mediates a change in glutamine metabolism in human MDA-MB-231 cancer cells.

<p>(<b>A)</b> Glutamine, glutamate and ammonia levels were analyzed in culture media of MDA-MB-231 cells. Glutamine anaplerosis was determined based on glutamine uptake and glutamate secretion. Both glutamine anaplerosis and the released ammonia were calculated as pmol*cell^-1*h^-1. (<b>B-C</b>) MIDs of target metabolites from [U¹³C₅]glutamine were evaluated in MDA-MB-231 cells after 56h of culture. Fraction of glutamine derived isotopologues (B) and contribution of [U¹³C₅]glutamine to target metabolites (C) were determined. In panel B, x-axis represents the mass isotopomer, while the y-axis indicates the relative abundance from [U¹³C₅]glutamine. (<b>D</b>) -/+ FSK cells were treated with BPTES for 24h and counted at 72h of culture. Percentage of reduction after the treatment is shown. All data represent the average of different experiments (n≥3).</p

The inhibition of PKA impacts on c-Src phosphorylation, mitochondrial respiration and intracellular ROS levels in MDA-MB-231 suspended cells.

<p>All analyses were performed in floating cells collected at 72h of culture in LG. <b>(A)</b> After 9h of treatment with DMSO or 10μM H89, Src and CREB phosphorylation was analyzed by Western blot. <b>(B)</b> After treatment with 1μM Saracatinib for 9h, trypan blue exclusion assay was performed. <b>(C)</b> OCR was measured with the Seahorse instrument. After cell treatment with DMSO or 10μM H89, the OCR of -/+H89 cells was measured every 30 minutes (steps 1–4, two measurements at each step). OCR was then measured after addition of 1μM FCCP (step 5, the last three measurements) to evaluate the mitochondrial reserve capacity (right histogram). The total duration of the analysis was 3–3.5h. The data are represented as mean±SEM. <b>(D)</b> Intracellular ATP level was measured in floating cells treated with DMSO or H89 for 3h. <b>(E)</b> Intracellular ROS were measured by using H₂DCFDA in floating cells treated with DMSO or H89 for 3–6h. <b>(F)</b> Viable count by trypan blue was performed in floating cells treated with 10μM H89 and 5mM NAC for 6h. All data represent the average of at least three independent experiments. *p<0.05, **p<0.01, ***p<0.001 Student’s t-test. <b>(G)</b> Schematic representation of the PKA-mediated resistance to glucose deprivation and <i>anoikis</i> in cancer cells.</p

PKA pathway induction results in autophagy activation in Transformed cells.

<p>All analyses were performed in Transformed cells cultured for 96h in LG, -/+ FSK. (<b>A)</b> MDC staining was performed in viable cells, while fixed cells were stained with a specific antibody against LC3. The images were visualized at 60X magnification. Scale bar 10μm. (<b>B)</b> Trypan blue exclusion assay was performed in cells -/+ FSK, treated or not with CQ at the indicated concentration for the last 24h of culture. Data are plotted as fold change over the equivalent control sample (- CQ). (<b>C)</b> Expression level of Grp78 and CHOP proteins was analyzed by Western blot in cells -/+ FSK and -/+ CQ with densitometric values. Eif2α expression was used as normalization. All data represent the average of different experiments (n>3).</p

MDA-MB-231 cells cultivation in LG results in a strong endogenous activation of PKA pathway with pro-survival effects.

<p>(<b>A)</b> PKA time-dependent activation in HG and LG was evaluated by Western blot analysis of p-(Ser/Thr) PKA substrates and pCREB S133. (<b>B)</b> Percentage of floating and dead cells at indicated time points of culture in LG. (<b>C)</b> At 72h of culture in LG suspended and adherent cells were separately collected to analyze PKA activity by Western blot as well as by ELISA assay. (<b>D-J)</b> All analyses were performed on floating cells collected at 72h of culture in LG. <b>(D)</b> Co-staining with a fluorescent substrate for active caspase 3, a p-PKAs antibody and DAPI. <b>(E)</b> PKA activity was determined by ELISA assay in cells treated or not treated with 10μM H89 for 6h. (<b>F)</b> Western blot analysis of pCREB S133 and p-(Ser/Thr) PKA substrates as well as of active caspase 3 in cells treated or not with H89. (<b>G</b>) Trypan blue exclusion assay was performed in cells treated or not treated with 10μM H89. (<b>H)</b> Colony formation assay was performed with cells treated or not with H89 for 6h. The colony formation was evaluated after 14 days of culture in HG as crystal violet absorbance. (<b>I-J)</b> Viable count by trypan blue was performed in cells treated with 10μM BPTES (I) or 10μM CQ (J) for 6h. All data represent the average of different experiments (n = 3).</p