Additional file 2: Figure S2. of Sidedness is prognostic in locoregional colon cancer: an analysis of 9509 Australian patients

Effect of adjuvant chemotherapy on overall survival in patients with left sided colon cancer. Description: Overall survival in patients with left sided colon cancer by receipt of adjuvant chemotherapy (n = 2030). (TIFF 40 kb

uPA contributes to bacterial disease dissemination <i>in vivo</i>.

<p><b>A</b> Cohorts of 10 age and sex matched mice were subcutaneously infected with 1×10⁹ CFU of GAS strain 5448 or 5448Δ<i>ska</i>. C57 black/6J <i>uPA −/−</i> mice (dashed line) showed a significant increase in survival (<i>P</i><0.05) compared to C57 black/6J mice (solid line). <b>B</b> Mouse uPA can mediate activation of human plasminogen, and human plasmin acquisition by 5448 (black fill) and 5448Δ<i>ska</i> (no fill). <b>C </b><i>AlbPLG1/uPA −/−</i> mice infected with 5448 (dashed line) or 5448Δ<i>ska</i> (dotted line) showed a significant increase in survival (<i>P</i><0.01) compared to <i>AlbPLG1</i> mice infected with 5448 (solid line). Survival data is combined from two independent experiments (<i>n</i> = 20), and significance was determined by log-rank test. Plasmin acquisition data is representative of two independent experiments, error bars indicate SEM (<i>n</i> = 3). Asterisks indicate statistical significance as determined by unpaired two-tailed students t-test, <i>P</i><0.05 (*), <i>P</i><0.001 (***).</p

Bacterial dissemination <i>in vivo</i> correlates with uPA mediated plasmin acquisition.

<p><b>A</b> Bacterial counts in the bloodstream and spleen of mice were significantly higher in <i>AlbPLG1</i> mice (black circles) than in <i>AlbPLG1/uPA −/−</i> mice (white circles). <b>B </b><i>AlbPLG1</i> and <i>AlbPLG1/uPA −/−</i> mice develop equivalent lesions 3 days post-inoculation with GAS strain 5448. <b>C</b> GAS strains 5448 (black fill) and 5448Δ<i>ska</i> (no fill) accumulate significantly lower levels of cell surface plasmin <i>ex vivo</i> in <i>AlbPLG1/uPA−/−</i> compared with <i>AlbPLG1</i> plasma. Dissemination and lesion size data is combined from 3 independent experiments (<i>n</i> = 6). Plasmin acquisition data is representative of two independent experiments, error bars indicate SEM (<i>n</i> = 3). Asterisks indicate statistical significance as determined by unpaired two-tailed students t-test, <i>P</i><0.05 (*), <i>P</i><0.001.</p

GAS facilitates enhanced uPA activity in plasma.

<p>Endogenous uPA activity in human plasma was measured using the uPA specific fluorogenic substrate Z-Gly-Gly-Arg-AMC, in the presence or absence of GAS strains 5448, 5448* and 5448Δ<i>ska</i>. Data is representative of four independent experiments, performed in duplicate. Background fluorescence has been subtracted from all values.</p

SerpinB2 modulates UPS activity and binds Ubiquitin.

<p><b>A.</b> Dose response of UPS activity (GFPu fluorescence) in wild-type (WT) and SerpinB2^-/- MEFs after 24 h treatment with proteasome inhibitor MG132. Note that fluorescence levels prior to addition of MG132 were similar in both cell types indicating similar degree of transfection with GFPu plasmid. Data represent mean fluorescence (_{515_20 blue}) ± SEM (n = 3). * Values for SerpinB2^-/- MEFS significantly different from wild-type cells at the corresponding MG132 concentration, <i>P</i> < 0.01; <b>B.</b> Immunoblot analysis validating correct expression of GFP and BiFC fusion proteins. GFP-SerpinB2, V1-SerpinB2 and SerpinB2-V1, and V2-SerpinB2 and SerpinB2-V2 were detected at the expected combined molecular weights of ~ 75 kDa, ~ 60 kDa (V1 is ~ 13 kDa), and ~ 55 kDa, (V2 is ~ 8 kDa), respectively; <b>C.</b> Epifluorescence microscopy showing GFP-SerpinB2 expression (green) in HEK293T cells, cell nuclei stained with DAPI (blue); <b>D.</b> Confocal microscopy showing BiFC signal (green) indicating SerpinB2 interaction with Ubiquitin (Ub) in HEK293T cells, cell nuclei stained with DAPI (blue). Scale bars = 20μm; <b>E.</b> GFP-SerpinB2 pull down and immunoblot analysis. GFP-SerpinB2 expressing HEK 293T cells were lysed (lysate) and samples applied to GFP affinity resin. Aliquots from the lysate and non-bound (nb) and eluted (elution) fractions (~ 40 μg) were analysed by immunoblot using antibodies against either SerpinB2 (α-SerpinB2) or Ub (α-Ub) as shown. Arrow indicates position of GFP-SerpinB2 in the SerpinB2 blot (left panel). A band of similar size is not present in the Ub blot (right panel). No proteins were detected in the elution fractions derived from untransfected or GFP only expressing cell lysates (data not shown); <b>F.</b> Dose response of autophagy activity (cellular associated Lyso-ID fluorescence) in WT and SerpinB2^-/- MEFs after 18 h treatment with autophagy inhibitor 3-MA or autophagy inducer verapamil. Data represent mean cellular fluorescence ± SEM (n = 3). **** indicates <i>P</i> < 0.0001; *** indicates <i>P</i> < 0.001, * indicates <i>P</i> < 0.05.</p

SerpinB2 protects cells from Htt Exon1 polyQ expansion-induced toxicity.

<p><b>A, B.</b> Viability of wild-type (A) or SerpinB2^-/- (B) MEFs following transient transfection with Htt_ex125Q-mCherry, Htt_ex146Q-mCherry, or mCherry expression alone (control) vectors. Data represent mean percentage of viable cells (as measured by SytoxRed exclusion and flow cytometry) normalised to mCherry only controls (n = 3 ± SEM). * Htt25q and Htt46q values significantly different from mCherry at 48 h post transfection, <i>P</i> < 0.01; <b>C.</b> Inclusion formation in wild-type versus SerpinB2^-/- MEFs 48 h post transfection with either Htt_ex125Q-mCherry or Htt_ex146Q-mCherry. Two types of Htt foci were observed, small (< 2um; white arrow heads) and large (> 2 um; white arrows); <b>D.</b> Distribution of Htt in MEFs was quantified as diffuse, small foci, or inclusion in each cell; <b>E, F, G.</b> Viability of wild-type (WT) (E) or SerpinB2^-/- (F) MEFS transduced with pMIG control empty vector (vector), or SerpinB2^-/- MEFS transduced with pMIG-SerpinB2 vector (WT rescue) (G) following transfection with Htt_ex125Q-mCherry, Htt_ex146Q-mCherry or mCherry expression vectors. Data represent mean percentage of viable cells normalised to mCherry only controls (n = 3 ± SEM). * Htt46Q value significantly different from mCherry at 48 h post transfection, <i>P</i> = 0.011.</p

SerpinB2 prevents beta-amyloid fibril formation <i>in vitro</i>.

<p><b>A.</b> Aβ_1–42 aggregation was followed by changes in thioflavin-T fluorescence (490 nm) over time in the absence (control) or presence of SerpinB2, SOD1 (negative control) or αB-crystallin (positive control) at a 1:10 final molar ratio of proteins:Aβ_1–42. Data represent mean fluorescence intensity with background controls subtracted (n = 2; representative experiment shown); <b>B–E</b>. Electron microscopy images of end point aggregates from Aβ_1–42 incubated alone (B), with SOD1 (C), SerpinB2 (D), or αB-crystallin (E). (scale bar = 200μm).</p

SerpinB2 binds misfolded proteins and suppresses BSA aggregation <i>in vitro</i>.

<p><b>A–C.</b> Dose dependent binding of SerpinB2 to native and misfolded proteins was determined using ELISA. Data represent mean absorbance (A₄₉₀) ± SEM (n = 3); <b>D.</b> Real-time turbidity assay of DTT-induced BSA aggregation in the absence or presence of SerpinB2, αB-crystallin or SerpinB14 (w/w ratio). Data represent mean absorbance (A₄₉₀) ± SEM (n = 3).</p

5′-Substituted Amiloride Derivatives as Allosteric Modulators Binding in the Sodium Ion Pocket of the Adenosine A_2A Receptor

The sodium ion site is an allosteric site conserved among many G protein-coupled receptors (GPCRs). Amiloride <b>1</b> and 5-(<i>N</i>,<i>N</i>-hexamethylene)­amiloride <b>2</b> (HMA) supposedly bind in this sodium ion site and can influence orthosteric ligand binding. The availability of a high-resolution X-ray crystal structure of the human adenosine A_2A receptor (hA_2AAR), in which the allosteric sodium ion site was elucidated, makes it an appropriate model receptor for investigating the allosteric site. In this study, we report the synthesis and evaluation of novel 5′-substituted amiloride derivatives as hA_2AAR allosteric antagonists. The potency of the amiloride derivatives was assessed by their ability to displace orthosteric radioligand [³H]­4-(2-((7-amino-2-(furan-2-yl)-[1,2,4]­triazolo­[1,5-<i>a</i>]-[1,3,5]­triazin-5-yl)­amino)­ethyl)­phenol ([³H]­ZM-241,385) from both the wild-type and sodium ion site W246A mutant hA_2AAR. 4-Ethoxy­phenethyl-substituted amiloride <b>12l</b> was found to be more potent than both amiloride and HMA, and the shift in potency between the wild-type and mutated receptor confirmed its likely binding to the sodium ion site

Image_3_Metastatic phenotype and immunosuppressive tumour microenvironment in pancreatic ductal adenocarcinoma: Key role of the urokinase plasminogen activator (PLAU).jpeg

BackgroundPrevious studies have revealed the role of dysregulated urokinase plasminogen activator (encoded by PLAU) expression and activity in several pathways associated with cancer progression. However, systematic investigation into the association of PLAU expression with factors that modulate PDAC (pancreatic ductal adenocarcinoma) progression is lacking, such as those affecting stromal (pancreatic stellate cell, PSC)-cancer cell interactions, tumour immunity, PDAC subtypes and clinical outcomes from potential PLAU inhibition.MethodsThis study used an integrated bioinformatics approach to identify prognostic markers correlated with PLAU expression using different transcriptomics, proteomics, and clinical data sets. We then determined the association of dysregulated PLAU and correlated signatures with oncogenic pathways, metastatic phenotypes, stroma, immunosuppressive tumour microenvironment (TME) and clinical outcome. Finally, using an in vivo orthotopic model of pancreatic cancer, we confirmed the predicted effect of inhibiting PLAU on tumour growth and metastasis.ResultsOur analyses revealed that PLAU upregulation is not only associated with numerous other prognostic markers but also associated with the activation of various oncogenic signalling pathways, aggressive phenotypes relevant to PDAC growth and metastasis, such as proliferation, epithelial-mesenchymal transition (EMT), stemness, hypoxia, extracellular cell matrix (ECM) degradation, upregulation of stromal signatures, and immune suppression in the tumour microenvironment (TME). Moreover, the upregulation of PLAU was directly connected with signalling pathways known to mediate PSC-cancer cell interactions. Furthermore, PLAU upregulation was associated with the aggressive basal/squamous phenotype of PDAC and significantly reduced overall survival, indicating that this subset of patients may benefit from therapeutic interventions to inhibit PLAU activity. Our studies with a clinically relevant orthotopic pancreatic model showed that even short-term PLAU inhibition is sufficient to significantly halt tumour growth and, importantly, eliminate visible metastasis.ConclusionElevated PLAU correlates with increased aggressive phenotypes, stromal score, and immune suppression in PDAC. PLAU upregulation is also closely associated with the basal subtype type of PDAC; patients with this subtype are at high risk of mortality from the disease and may benefit from therapeutic targeting of PLAU.</p