CD73 facilitates EMT progression and promotes lung metastases in triple-negative breast cancer

CD73 is a cell surface ecto-5 ' -nucleotidase, which converts extracellular adenosine monophosphate to adenosine. High tumor CD73 expression is associated with poor outcome among triple-negative breast cancer (TNBC) patients. Here we investigated the mechanisms by which CD73 might contribute to TNBC progression. This was done by inhibiting CD73 with adenosine 5 '-(alpha, beta -methylene) diphosphate (APCP) in MDA-MB-231 or 4T1 TNBC cells or through shRNA-silencing (sh-CD73). Effects of such inhibition on cell behavior was then studied in normoxia and hypoxia in vitro and in an orthotopic mouse model in vivo. CD73 inhibition, through shRNA or APCP significantly decreased cellular viability and migration in normoxia. Inhibition of CD73 also resulted in suppression of hypoxia-induced increase in viability and prevented cell protrusion elongation in both normoxia and hypoxia in cancer cells. Sh-CD73 4T1 cells formed significantly smaller and less invasive 3D organoids in vitro, and significantly smaller orthotopic tumors and less lung metastases than control shRNA cells in vivo. CD73 suppression increased E-cadherin and decreased vimentin expression in vitro and in vivo, proposing maintenance of a more epithelial phenotype. In conclusion, our results suggest that CD73 may promote early steps of tumor progression, possibly through facilitating epithelial-mesenchymal transition

Documentation of protein crystallization techniques and characterization of crystals

X-ray crystallography is a method for determining the detailed three-dimensional structure of proteins and protein complexes. It is based on the diffraction of X-rays from protein crystals. The principle is to grow suitable protein crystals and shoot them with an intense X-ray beam. The resulting scattering of X-rays is recorded and the resulting data can be used in molecular modeling. The models can be used for example in structure-based drug design and site-directed mutagenesis. This thesis project was conducted in the Turku Center for Biotechnology (CBT), which is a joint department of the University of Turku and Åbo Akademi University. It provides high-end technologies and expertise to academic and industrial researchers. The purpose of this thesis was to document the methods used in X-ray crystallography in CBT and to produce easy-to-read protocols for the staff.Röntgenkristallografia on yleinen tekniikka, jolla voidaan selvittää proteiinien ja proteiinikompleksien kolmiulotteinen rakenne. Se perustuu röntgensäteiden sirontaan proteiineista muodostuvien kiteiden sisältämien atomien elektroneista. Periaatteena on ensin kasvattaa sopiva proteiinikide, johon kohdistetaan intensiivinen röntgensäde. Säteiden sironta mitataan ja saatua dataa käytetään molekyylin rakennetutkimuksessa. Tuotettuja malleja voidaan käyttää apuna esim. lääkkeiden suunnittelussa ja paikkakohtaisessa mutageneesissä. Tämä opinnäyte tehtiin Turun biotekniikan keskuksessa (BTK), joka on Turun yliopiston ja Åbo akademin yhteinen erillislaitos. BTK tarjoaa korkean tason tekniikkaa ja osaamista akateemisille ja teollisen alan tutkijoille. Työn tarkoituksena oli dokumentoida biotekniikan keskuksen käyttämät tekniikat röntgenkristallografiassa ja kirjoittaa niiden pohjalta protokollat henkilökunnalle

Exposure to per- and polyfluoroalkyl substances in early pregnancy and risk of cerebral palsy in children

BACKGROUND: Most cerebral palsy (CP) cases have an unexplained etiology, but a role for environmental exposures has been suggested. One purported environmental risk factor is exposure to endocrine-disrupting pollutants specifically per- and polyfluoroalkyl substances (PFAS).OBJECTIVES: We investigated the association between prenatal PFAS exposures and CP in Swedish children.METHODS: In this case-control study, 322 CP cases, 343 population controls, and 258 preterm controls were identified from a birth registry in combination with a CP follow-up program from 1995 to 2014 and linked to a biobank which contains serum samples from week 10-14 of pregnancy. Maternal serum concentrations of four PFAS compounds: perfluorohexane sulfonate (PFHxS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and perfluorooctane sulfonate (PFOS) were analyzed using liquid chromatography-tandem-mass-spectrometry. We estimated odds ratios (ORs) and 95 % confidence intervals (CIs) for CP and each PFAS in quartiles and as continuous variables controlling for various sociodemographic and lifestyle factors.RESULTS: In crude and adjusted analyses, we did not find consistent evidence of associations between serum PFHxS, PFOA, PFNA, PFOS and concentrations in early pregnancy and CP, except in preterm infants. The ORs comparing the highest PFAS quartiles to the lowest were 1.05 (95 % CI: 0.63-1.76), 0.96 (95 % CI: 0.55-1.68), 0.71 (95 % CI: 0.41-1.25), and 1.17 (95 % CI: 0.61-2.26), for PFHxS, PFOA, PFNA, and PFOS, respectively. Some positive associations were observed for preterm infants, but the results were imprecise. Similar patterns were observed in analyses treating PFAS as continuous variables.CONCLUSIONS: In this study, we found little evidence that early pregnancy prenatal exposure to PFHxS, PFOA, PFNA, or PFOS increases the risk of CP. However, some positive associations were observed for preterm cases and warrant further investigation

Evaluation of the anticancer activity of RIN-1, a Notch signaling modulator, in head and neck squamous cell carcinoma

Abstract Notch signalling is one of the key molecular pathways involved in cell-to-cell signal transduction. Although the mechanisms of action of the NOTCH receptors are already relatively well known, their biological implications remain unclear, especially during the initiation and progression of head and neck squamous cell carcinoma (HNSCC). Here, we present the growth- and differentiation-modulating effects of various “next generation” small molecule Notch modulators represented by RIN-1, and CB-103, on HNSCC, compared to gamma secretase inhibitors as “conventional” NOTCH interfering compounds, like DAPT. These molecules were tested in different cell- and tissue culture conditions represented by 2D monolayer, non-adherent or spheroid culture, 3D organoid cultures, and zebrafish in vivo model. The most pronounced, pleiotropic effects were observed for the NOTCH modulator RIN-1. At the molecular level, RIN-1-dependent activation of Notch signalling led to characteristic changes in the expression of NOTCH-regulated targets, i.e., the transcriptional suppressors HES1 and HEY1, p21 (CDKN1A) cell cycle inhibitor, and pro-apoptotic BAX markers. These changes led to restriction of proliferation, growth, and reduced motility of HNSCC cells in 2D cultures. Consequently, cell cycle arrest in the G2-M phase and induction of apoptosis were observed. Similar anticancer effects were observed in 3D cultures and in the zebrafish model. In contrast, RIN-1 treatment resulted in inhibition of Notch signalling and the growth of HNSCC spheroids under non-adherent cell culture conditions. Our results suggest that modulation of Notch signalling could be used as a chemotherapeutic agent in selected patients with intact NOTCH signaling

CD73 facilitates EMT progression and promotes lung metastases in triple-negative breast cancer

CD73 is a cell surface ecto-5′-nucleotidase, which converts extracellular adenosine monophosphate to adenosine. High tumor CD73 expression is associated with poor outcome among triple-negative breast cancer (TNBC) patients. Here we investigated the mechanisms by which CD73 might contribute to TNBC progression. This was done by inhibiting CD73 with adenosine 5′-(α, β-methylene) diphosphate (APCP) in MDA-MB-231 or 4T1 TNBC cells or through shRNA-silencing (sh-CD73). Effects of such inhibition on cell behavior was then studied in normoxia and hypoxia in vitro and in an orthotopic mouse model in vivo. CD73 inhibition, through shRNA or APCP significantly decreased cellular viability and migration in normoxia. Inhibition of CD73 also resulted in suppression of hypoxia-induced increase in viability and prevented cell protrusion elongation in both normoxia and hypoxia in cancer cells. Sh-CD73 4T1 cells formed significantly smaller and less invasive 3D organoids in vitro, and significantly smaller orthotopic tumors and less lung metastases than control shRNA cells in vivo. CD73 suppression increased E-cadherin and decreased vimentin expression in vitro and in vivo, proposing maintenance of a more epithelial phenotype. In conclusion, our results suggest that CD73 may promote early steps of tumor progression, possibly through facilitating epithelial–mesenchymal transition.publishedVersionPeer reviewe

Validation of novel biomarkers for prostate cancer progression by the combination of bioinformatics, clinical and functional studies

The identification and validation of biomarkers for clinical applications remains an important issue for improving diagnostics and therapy in many diseases, including prostate cancer. Gene expression profiles are routinely applied to identify diagnostic and predictive biomarkers or novel targets for cancer. However, only few predictive markers identified in silico have also been validated for clinical, functional or mechanistic relevance in disease progression. In this study, we have used a broad, bioinformatics-based approach to identify such biomarkers across a spectrum of progression stages, including normal and tumor-adjacent, premalignant, primary and late stage lesions. Bioinformatics data mining combined with clinical validation of biomarkers by sensitive, quantitative reverse-transcription PCR (qRT-PCR), followed by functional evaluation of candidate genes in disease-relevant processes, such as cancer cell proliferation, motility and invasion. From 300 initial candidates, eight genes were selected for validation by several layers of data mining and filtering. For clinical validation, differential mRNA expression of selected genes was measured by qRT-PCR in 197 clinical prostate tissue samples including normal prostate, compared against histologically benign and cancerous tissues. Based on the qRT-PCR results, significantly different mRNA expression was confirmed in normal prostate versus malignant PCa samples (for all eight genes), but also in cancer-adjacent tissues, even in the absence of detectable cancer cells, thus pointing to the possibility of pronounced field effects in prostate lesions. For the validation of the functional properties of these genes, and to demonstrate their putative relevance for disease-relevant processes, siRNA knock-down studies were performed in both 2D and 3D organotypic cell culture models. Silencing of three genes (DLX1, PLA2G7 and RHOU) in the prostate cancer cell lines PC3 and VCaP by siRNA resulted in marked growth arrest and cytotoxicity, particularly in 3D organotypic cell culture conditions. In addition, silencing of PLA2G7, RHOU, ACSM1, LAMB1 and CACNA1D also resulted in reduced tumor cell invasion in PC3 organoid cultures. For PLA2G7 and RHOU, the effects of siRNA silencing on proliferation and cell-motility could also be confirmed in 2D monolayer cultures. In conclusion, DLX1 and RHOU showed the strongest potential as useful clinical biomarkers for PCa diagnosis, further validated by their functional roles in PCa progression. These candidates may be useful for more reliable identification of relapses or therapy failures prior to the recurrence local or distant metastases

Correction: Validation of Novel Biomarkers for Prostate Cancer Progression by the Combination of Bioinformatics, Clinical and Functional Studies.

[This corrects the article DOI: 10.1371/journal.pone.0155901.]

Images of 3D organotypic cell cultures of PC3 cells, embed in Matrigel, after segmentation and subsequent image analysis using the AMIDA software package.

<p>A. Untreated cells form large organoids with overt invasive processes. B. Silencing of PLA2G7 and C. laminin beta 1 (LMNB1) result in well-rounded, poorly invasive organoids, with higher cell density. D. In contrast, silencing of the TDRD1 gene results in significant induction of invasive properties, further loss of the structural organization or maturation of organoids, and decreased cell density.</p

ROC analyses for KLK3 mRNA levels and expression levels of 8 target mRNAs in cases classified as positive or negative for PCa.

<p>To simplify the analysis, patients with two cancerous/benign samples were represented by a single value of mRNA expression of each gene. A. RP-PCa samples were considered as positive samples and compared against all CP samples (defined as negative). High sensitivity and specificity was observed for all eight biomarkers. B. RP-Be samples were considered as positive samples and compared against all CP samples (defined as negative).</p