Urine biomarkers enable pancreatic cancer detection up to 2 years before diagnosis.

The poor prognosis of pancreatic ductal adenocarcinoma (PDAC) is mainly attributed to late diagnosis. We assessed the predictive performance of our previously reported urine biomarker panel for earlier detection of PDAC (LYVE1, REG1B and TFF1) in prediagnostic samples, alone and in combination with plasma CA19-9. This nested case-control study included 99 PDAC cases with urine samples prospectively collected up to 5 years prior to PDAC diagnosis and 198 matched controls. The samples were obtained from the Shanghai Women's Health Study (SWHS), the Shanghai Men's Health Studies (SMHS) and the Southern Community Cohort Study (SCCS). The urine biomarkers were measured by ELISA. Plasma CA19-9 was quantified by Luminex. Multiple logistic regression and Wilcoxon rank-sum and Mann-Whitney test were used for analysis. The internal validation approach was applied and the validated AUC estimators are reported on. The algorithm of urinary protein panel, urine creatinine and age named PancRISK, displayed similar AUC as CA19-9 up to 1 year before PDAC diagnosis (AUC = 0.79); however, the combination enhanced the AUCs to 0.89, and showed good discriminative ability (AUC = 0.77) up to 2 years. The combination showed sensitivity (SN) of 72% at 90% specificity (SP), and SP of 59% at 90% SN up to 1 year and 60% SN with 80% SP and 53% SP with 80% SN up to 2 years before PDAC diagnosis. Adding the clinical information on BMI value resulted in the overall improvement in performance of the PancRISK score. When combined with CA19-9, the urinary panel reached a workable model for detecting PDAC cases up to 2 years prior to diagnosis.

The anti-psychotic drug pimozide is a novel chemotherapeutic for breast cancer.

Pimozide, an antipsychotic drug of the diphenylbutylpiperidine class, has been shown to suppress cell growth of breast cancer cells in vitro. In this study we further explore the inhibitory effects of this molecule in cancer cells. We found that Pimozide inhibited cell proliferation in a dose- and time-dependent manner in MDA-MB-231 breast cancer cells and A549 lung cancer cells. Furthermore, we found that Pimozide also promoted apoptosis as demonstrated by cell cycle arrest and induction of double-strand DNA breaks but did not result in any effect in the non-transformed MCF10A breast cell line. In order to shed new lights into the molecular pathways affected by Pimozide, we show that Pimozide downregulated RAN GTPase and AKT at both protein and mRNA levels and inhibited the AKT signaling pathway in MDA-MB-231 breast cancer cells. Pimozide also inhibited the epithelial mesenchymal transition and cell migration and downregulated the expression of MMPs. Administration of Pimozide showed a potent in vivo antitumor activity in MDA-MB-231 xenograft animal model and reduced the number of lung metastases by blocking vascular endothelial growth factor receptor 2. Furthermore, Pimozide inhibited myofibroblast formation as evaluated by the reduction in α-smooth muscle actin containing cells. Thus, Pimozide might inhibit tumor development by suppressing angiogenesis and by paracrine stimulation provided by host reactive stromal cells. These results demonstrate a novel in vitro and in vivo antitumor activity of Pimozide against breast and lung cancer cells and provide the proof of concept for a putative Pimozide as a novel approach for cancer therapy.

ΔNp63γ/SRC/Slug Signaling Axis Promotes Epithelial-to-Mesenchymal Transition in Squamous Cancers.

Purpose: To investigate the regulation of epithelial-to-mesenchymal transition (EMT) in head and neck squamous cell carcinoma (HNSCC) and its importance in tumor invasion.Experimental Design: We use a three-dimensional invasive organotypic raft culture model of human foreskin keratinocytes expressing the E6/E7 genes of the human papilloma virus-16, coupled with bioinformatic and IHC analysis of patient samples to investigate the role played by EMT in invasion and identify effectors and upstream regulatory pathways.Results: We identify SNAI2 (Slug) as a critical effector of EMT-activated downstream of TP63 overexpression in HNSCC. Splice-form-specific depletion and rescue experiments further identify the ΔNp63γ isoform as both necessary and sufficient to activate the SRC signaling axis and SNAI2-mediated EMT and invasion. Moreover, elevated SRC levels are associated with poor outcome in patients with HNSCC in The Cancer Genome Atlas dataset. Importantly, the effects on EMT and invasions and SNAI2 expression can be reversed by genetic or pharmacologic inhibition of SRC.Conclusions: Overexpression of ΔNp63γ modulates cell invasion by inducing targetable SRC-Slug-evoked EMT in HNSCC, which can be reversed by inhibitors of the SRC signaling. Clin Cancer Res; 24(16); 3917-27. ©2018 AACR.

Dual effects of radiation bystander signaling in urothelial cancer: purinergic-activation of apoptosis attenuates survival of urothelial cancer and normal urothelial cells.

Radiation therapy (RT) delivers tumour kill, directly and often via bystander mechanisms. Bladder toxicity is a dose limiting constraint in pelvic RT, manifested as radiation cystitis and urinary symptoms. We aimed to investigate the impact of radiation-induced bystander signaling on normal/cancer urothelial cells. Human urothelial cancer cells T24, HT1376 and normal urothelial cells HUC, SV-HUC were used. Cells were irradiated and studied directly, or conditioned medium from irradiated cells (CM) was transferred to naïve, cells. T24 or SV-HUC cells in the shielded half of irradiated flasks had increased numbers of DNA damage foci vs non-irradiated cells. A physical barrier blocked this response, indicating release of transmitters from irradiated cells. Clonogenic survival of shielded T24 or SV-HUC was also reduced; a physical barrier prevented this phenomenon. CM-transfer increased pro-apoptotic caspase-3 activity, increased cleaved caspase-3 and cleaved PARP expression and reduced survival protein XIAP expression. This effect was mimicked by ATP. ATP or CM evoked suramin-sensitive Ca2+-signals. Irradiation increased [ATP] in CM from T24. The CM-inhibitory effect on T24 clonogenic survival was blocked by apyrase, or mimicked by ATP. We conclude that radiation-induced bystander signaling enhances urothelial cancer cell killing via activation of purinergic pro-apoptotic pathways. This benefit is accompanied by normal urothelial damage indicating RT bladder toxicity is also bystander-mediated.

p63 drives invasion in keratinocytes expressing HPV16 E6/E7 genes through regulation of Src-FAK signalling.

Using microarray information from oro-pharyngeal data sets and results from primary human foreskin keratinocytes (HFK) expressing Human Papilloma Virus (HPV)-16 E6/E7 proteins, we show that p63 expression regulates signalling molecules which initiate cell migration such as Src and focal adhesion kinase (FAK) and induce invasion in 3D-organotypic rafts; a phenotype that can be reversed by depletion of p63. Knockdown of Src or FAK in the invasive cells restored focal adhesion protein paxillin at cell periphery and impaired the cell migration. In addition, specific inhibition of FAK (PF573228) or Src (dasatinib) activities mitigated invasion and attenuated the expression/activity of matrix metalloproteinase 14 (MMP14), a pivotal MMP in the MMP activation cascade. Expression of constitutively active Src in non-invasive HFK expressing E6/E7 proteins upregulated the activity of c-Jun and MMP14, and induced invasion in rafts. Depletion of Src, FAK or AKT in the invasive cells normalised the expression/activity of c-Jun and MMP14, thus implicating the Src-FAK/AKT/AP-1 signalling in MMP14-mediated extra-cellular matrix remodelling. Up-regulation of Src, AP-1, MMP14 and p63 expression was confirmed in oro-pharyngeal cancer. Since p63 transcriptionally regulated expression of many of the genes in this signalling pathway, it suggests that it has a central role in cancer progression.

Attenuation of urokinase activity during experimental ischaemia protects the cerebral barrier from damage through regulation of matrix metalloproteinase-2 and NAD(P)H oxidase.

Ischaemic injury impairs the integrity of the blood-brain barrier (BBB). In this study, we investigated the molecular causes of this defect with regard to the putative correlations among NAD(P)H oxidase, plasminogen-plasmin system components, and matrix metalloproteinases. Hence, the activities of NAD(P)H oxidase, matrix metalloproteinase-2, urokinase-type plasminogen activator (uPA), and tissue-type plasminogen activator (tPA), and superoxide anion levels, were assessed in human brain microvascular endothelial cells (HBMECs) exposed to oxygen-glucose deprivation (OGD) alone or OGD followed by reperfusion (OGD + R). The integrity of an in vitro model of BBB comprising HBMECs and astrocytes was studied by measuring transendothelial electrical resistance and the paracellular flux of albumin. OGD with or without reperfusion (OGD ± R) radically perturbed barrier function while concurrently enhancing uPA, tPA and NAD(P)H oxidase activities and superoxide anion release in HBMECs. Pharmacological inactivation of NAD(P)H oxidase attenuated OGD ± R-mediated BBB damage through modulation of matrix metalloproteinase-2 and tPA, but not uPA activity. Overactivation of NAD(P)H oxidase in HBMECs via cDNA electroporation of its p22-phox subunit confirmed the involvement of tPA in oxidase-mediated BBB disruption. Interestingly, blockade of uPA or uPA receptor preserved normal BBB function by neutralizing both NAD(P)H oxidase and matrix metalloproteinase-2 activities. Hence, selective targeting of uPA after ischaemic strokes may protect cerebral barrier integrity and function by concomitantly attenuating basement membrane degradation and oxidative stress.

Inhibition of Rho-kinase protects cerebral barrier from ischaemia-evoked injury through modulations of endothelial cell oxidative stress and tight junctions.

Ischaemic strokes evoke blood-brain barrier (BBB) disruption and oedema formation through a series of mechanisms involving Rho-kinase activation. Using an animal model of human focal cerebral ischaemia, this study assessed and confirmed the therapeutic potential of Rho-kinase inhibition during the acute phase of stroke by displaying significantly improved functional outcome and reduced cerebral lesion and oedema volumes in fasudil- versus vehicle-treated animals. Analyses of ipsilateral and contralateral brain samples obtained from mice treated with vehicle or fasudil at the onset of reperfusion plus 4 h post-ischaemia or 4 h post-ischaemia alone revealed these benefits to be independent of changes in the activity and expressions of oxidative stress- and tight junction-related parameters. However, closer scrutiny of the same parameters in brain microvascular endothelial cells subjected to oxygen-glucose deprivation ± reperfusion revealed marked increases in prooxidant NADPH oxidase enzyme activity, superoxide anion release and in expressions of antioxidant enzyme catalase and tight junction protein claudin-5. Cotreatment of cells with Y-27632 prevented all of these changes and protected in vitro barrier integrity and function. These findings suggest that inhibition of Rho-kinase after acute ischaemic attacks improves cerebral integrity and function through regulation of endothelial cell oxidative stress and reorganization of intercellular junctions. Inhibition of Rho-kinase (ROCK) activity in a mouse model of human ischaemic stroke significantly improved functional outcome while reducing cerebral lesion and oedema volumes compared to vehicle-treated counterparts. Studies conducted with brain microvascular endothelial cells exposed to OGD ± R in the presence of Y-27632 revealed restoration of intercellular junctions and suppression of prooxidant NADPH oxidase activity as important factors in ROCK inhibition-mediated BBB protection.

PKC-ß exacerbates in vitro brain barrier damage in hyperglycemic settings via regulation of RhoA/Rho-kinase/MLC2 pathway.

Stroke patients with hyperglycemia (HG) develop higher volumes of brain edema emerging from disruption of blood-brain barrier (BBB). This study explored whether inductions of protein kinase C-ß (PKC-ß) and RhoA/Rho-kinase/myosin-regulatory light chain-2 (MLC2) pathway may account for HG-induced barrier damage using an in vitro model of human BBB comprising human brain microvascular endothelial cells (HBMEC) and astrocytes. Hyperglycemia (25 mmol/L D-glucose) markedly increased RhoA/Rho-kinase protein expressions (in-cell westerns), MLC2 phosphorylation (immunoblotting), and PKC-ß (PepTag assay) and RhoA (Rhotekin-binding assay) activities in HBMEC while concurrently reducing the expression of tight junction protein occludin. Hyperglycemia-evoked in vitro barrier dysfunction, confirmed by decreases in transendothelial electrical resistance and concomitant increases in paracellular flux of Evan's blue-labeled albumin, was accompanied by malformations of actin cytoskeleton and tight junctions. Suppression of RhoA and Rho-kinase activities by anti-RhoA immunoglobulin G (IgG) electroporation and Y-27632, respectively prevented morphologic changes and restored plasma membrane localization of occludin. Normalization of glucose levels and silencing PKC-ß activity neutralized the effects of HG on occludin and RhoA/Rho-kinase/MLC2 expression, localization, and activity and consequently improved in vitro barrier integrity and function. These results suggest that HG-induced exacerbation of the BBB breakdown after an ischemic stroke is mediated in large part by activation of PKC-ß.

Current therapeutic strategies to mitigate the eNOS dysfunction in ischaemic stroke.

Impairment of endothelial nitric oxide synthase (eNOS) activity is implicated in the pathogenesis of endothelial dysfunction in many diseases including ischaemic stroke. The modulation of eNOS during and/or following ischaemic injury often represents a futile compensatory mechanism due to a significant decrease in nitric oxide (NO) bioavailability coupled with dramatic increases in the levels of reactive oxygen species that further neutralise NO. However, applications of a number of therapeutic agents alone or in combination have been shown to augment eNOS activity under a variety of pathological conditions by potentiating the expression and/or activity of Akt/eNOS/NO pathway components. The list of these therapeutic agents include NO donors, statins, angiotensin-converting enzyme inhibitors, calcium channel blockers, phosphodiesterase-3 inhibitors, aspirin, dipyridamole and ellagic acid. While most of these compounds exhibit anti-platelet properties and are able to up-regulate eNOS expression in endothelial cells and platelets, others suppress eNOS uncoupling and tetrahydrobiopterin (an eNOS stabiliser) oxidation. As the number of therapeutic molecules that modulate the expression and activity of eNOS increases, further detailed research is required to reveal their mode of action in preventing and/or reversing the endothelial dysfunction.

Small GTPase RhoA and its effector rho kinase mediate oxygen glucose deprivation-evoked in vitro cerebral barrier dysfunction.

BACKGROUND AND PURPOSE: Enhanced vascular permeability attributable to disruption of blood-brain barrier results in the development of cerebral edema after stroke. Using an in vitro model of the brain barrier composed of human brain microvascular endothelial cells and human astrocytes, this study explored whether small GTPase RhoA and its effector protein Rho kinase were involved in permeability changes mediated by oxygen-glucose deprivation (OGD), key pathological phenomena during ischemic stroke. METHODS: OGD increased RhoA and Rho kinase protein expressions in human brain microvascular endothelial cells and human astrocytes while increasing or unaffecting that of endothelial nitric oxide synthase in respective cells. Reperfusion attenuated the expression and activity of RhoA and Rho kinase in both cell types compared to their counterparts exposed to equal periods of OGD alone while selectively increasing human brain microvascular endothelial cells endothelial nitric oxide synthase protein levels. OGD compromised the barrier integrity as confirmed by decreases in transendothelial electric resistance and concomitant increases in flux of permeability markers sodium fluorescein and Evan's blue albumin across cocultures. Transfection of cells with constitutively active RhoA also increased flux and reduced transendothelial electric resistance, whereas inactivation of RhoA by anti-RhoA Ig electroporation exerted opposite effects. In vitro cerebral barrier dysfunction was accompanied by myosin light chain overphosphorylation and stress fiber formation. Reperfusion and treatments with a Rho kinase inhibitor Y-27632 significantly attenuated barrier breakdown without profoundly altering actin structure. CONCLUSIONS: Increased RhoA/Rho kinase/myosin light chain pathway activity coupled with changes in actin cytoskeleton account for OGD-induced endothelial barrier breakdown.