Mutations of the EPHB6 Receptor Tyrosine Kinase Induce a Pro-Metastatic Phenotype in Non-Small Cell Lung Cancer

Alterations of Eph receptor tyrosine kinases are frequent events in human cancers. Genetic variations of EPHB6 have been described but the functional outcome of these alterations is unknown. The current study was conducted to screen for the occurrence and to identify functional consequences of EPHB6 mutations in non-small cell lung cancer. Here, we sequenced the entire coding region of EPHB6 in 80 non-small cell lung cancer patients and 3 tumor cell lines. Three potentially relevant mutations were identified in primary patient samples of NSCLC patients (3.8%). Two point mutations led to instable proteins. An in frame deletion mutation (del915-917) showed enhanced migration and accelerated wound healing in vitro. Furthermore, the del915-917 mutation increased the metastatic capability of NSCLC cells in an in vivo mouse model. Our results suggest that EPHB6 mutations promote metastasis in a subset of patients with non-small cell lung cancer

Proteinase-Activated Receptor 1 (PAR1) Regulates Leukemic Stem Cell Functions

External signals that are mediated by specific receptors determine stem cell fate. The thrombin receptor PAR1 plays an important role in haemostasis, thrombosis and vascular biology, but also in tumor biology and angiogenesis. Its expression and function in hematopoietic stem cells is largely unknown. Here, we analyzed expression and function of PAR1 in primary hematopoietic cells and their leukemic counterparts. AML patients' blast cells expressed much lower levels of PAR1 mRNA and protein than CD34+ progenitor cells. Constitutive Par1-deficiency in adult mice did not affect engraftment or stem cell potential of hematopoietic cells. To model an AML with Par1-deficiency, we retrovirally introduced the oncogene MLL-AF9 in wild type and Par1−/− hematopoietic progenitor cells. Par1-deficiency did not alter initial leukemia development. However, the loss of Par1 enhanced leukemic stem cell function in vitro and in vivo. Re-expression of PAR1 in Par1−/− leukemic stem cells delayed leukemogenesis in vivo. These data indicate that Par1 contributes to leukemic stem cell maintenance

The Long Noncoding MALAT-1 RNA Indicates a Poor Prognosis in Non-small Cell Lung Cancer and Induces Migration and Tumor Growth

Introduction:The functions of large noncoding RNAs (ncRNAs) have remained elusive in many cases. Metastasis-Associated-in-Lung-Adenocarcinoma-Transcript-1 (MALAT-1) is an ncRNA that is highly expressed in several tumor types.Methods:Overexpression and RNA interference (RNAi) approaches were used for the analysis of the biological functions of MALAT-1 RNA. Tumor growth was studied in nude mice. For prognostic analysis, MALAT-1 RNA was detected on paraffin-embedded non-small cell lung cancer (NSCLC) tissue probes (n = 352) using in situ hybridization.Results:MALAT-1 was highly expressed in several human NSCLC cell lines. MALAT-1 expression was regulated by an endogenous negative feedback loop. In A549 NSCLCs, RNAi-mediated suppression of MALAT-1 RNA suppressed migration and clonogenic growth. Forced expression of MALAT-1 in NIH 3T3 cells significantly increased migration. Upon injection into nude mice, NSCLC xenografts with decreased MALAT-1 expression were impaired in tumor formation and growth. In situ hybridization on paraffin-embedded lung cancer tissue probes revealed that high MALAT-1 RNA expression in squamous cell carcinoma of the lung was associated with a poor prognosis. On genetic level, MALAT-1 displays the strongest association with genes involved in cancer like cellular growth, movement, proliferation, signaling, and immune regulation.Conclusions:These data indicate that MALAT-1 expression levels are associated with patient survival and identify tumor-promoting functions of MALAT-1

PAR1 expression in primary patient samples.

<p><b>4A</b>. Micrographs of Tissue Array analysis from NBM and AML patients stained with anti-PAR1 antibody and Fast-Red secondary antibody contrasted with hematoxylin and eosin. Overview (upper panel) and magnification of one example of CD34⁺ and AML samples that were defined PAR1-negative (lower left) and PAR1-positive (lower right). <b>4B</b>. Quantitative Tissue Array analysis of PAR1 expression using categories of staining intensity as positive or negative. Significantly more AML patient samples were negative for PAR1 expression than CD34⁺ healthy patient samples (p = 0.003, Chi-square test). <b>4C</b>. PAR1 protein was significantly less abundant in bone marrow cells from human Acute Myeloid Leukemia (AML) patients compared to CD34-positive bone marrow cells in Tissue Array samples. *p<0.05, Chi-square test.</p

PAR1 is expressed in hematopoietic cells.

<p><b>1A</b>. PAR1 was analyzed in mRNA microarray expression data from FACS sorted bone marrow cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094993#pone.0094993-Hebestreit1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094993#pone.0094993-Novershtern1" target="_blank">[23]</a>. Highest expression was found in hematopoietic stem cells (HSC) and cells of the erythroid/megakaryocyte and of the T-cell lineage. Shown here are log arbitrary units. <b>1B</b>. Left-hand side: To sort for the different murine bone marrow subpopulation, total bone marrow was stained with lineage-markers, sca1 and c-kit. Lineage-negative, sca1⁺, c-kit⁺ (LSK) cells were further divided into long-term (LT)-HSCs as Flt3⁻CD34⁻ population, short-term (ST)-HSCs as Flt3⁻CD34⁺ cells and multipotent progenitors (MPPs) as Flt3⁺CD34⁺ cells (upper panel). Common lymphoid progenitors (CLPs) were defined as lineage-negative, IL7R⁺c-kit⁺ cells. Upper and lower right panel: <i>Par1</i> mRNA expression was determined by real-time quantitative RT-PCR using cDNA from the FACS-sorted murine bone marrow subpopulations and Par1 expression was normalized to GAPDH expression. Par1 was expressed in all hematopoietic stem/progenitor subpopulations and CD3⁺ T-cells whereas monocytes/macrophages/granulocytes (CD11b⁺) or erythrocytic (Ter119⁺) or B-cells (B220⁺) expressed low or no Par1.</p

Absence of <i>Par1</i> accelerates MLL-AF9 driven murine leukemogenesis.

<p><b>5A</b>. Schematic overview about the performed transduction and transplantation experiments. Bone marrow isolated from <i>Par1^+/+</i> or <i>Par1^−/−</i> mice was retrovirally transduced with MLL-AF9/GFP. Equal numbers of positive cells were transplanted into lethally irradiated recipients, which were then subjected to different analyses and subsequent serial transplantations. <b>5B</b>. Survival curves of recipient mice which were transplanted with bone marrow cells of <i>Par1^+/+</i> or <i>Par1^−/−</i> mice that were retrovirally transduced with MLL-AF9 (n = 8 of each genotype). Cells of both genotypes led to a fatal leukemic disease with comparable latency. <b>5C</b>. Survival curves of secondary recipient mice which were transplanted with bone marrow cells of leukemic mice derived from the primary transplantation shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094993#pone-0094993-g005" target="_blank">Fig. 5B</a>. The secondary recipients of <i>Par1^−/−</i>;MLL-AF9 cells (n = 14) died after a significantly shorter latency than mice transplanted with <i>Par1^+/+</i>;MLL-AF9 primary blasts (n = 15; p<0.001). <b>5D</b>. The phenotypic analysis of blasts of the secondary leukemic mice did not reveal differences in CD11b expression between <i>Par1^+/+</i>;MLL-AF9 and <i>Par1^−/−</i>;MLL-AF9 cells. <b>5E</b>. <i>Par1^−/−</i>;MLL-AF9 transplanted mice (n = 8) exhibited a strong tendency towards higher percentages ofc-kit expressing cells in spleens (p = 0.055, t-test) and bone marrow (p = 0.22, t-test)compared to <i>Par1^+/+</i>;MLL-AF9 transplanted mice (n = 4).</p

Summary of non-synonymous mutations for EPHB6 (NM_004445 and NP_004436) found in tumors.

<p>Note: The table contains data from the databases of <a href="http://www.sanger.ac.uk/genetics/CGP/cosmic/" target="_blank">http://www.sanger.ac.uk/genetics/CGP/cosmic/</a>, <a href="http://strubiol.icr.ac.uk/extra/mokca" target="_blank">http://strubiol.icr.ac.uk/extra/mokca</a>, and the references were listed in the column of “Pubmed Id”. The NSCLC mutations identified in this study were marked as “not reported”. Two sequence homology-based tools were used to predict the potential impact of the identified non-synonymous substitutions on protein function: Sort Intolerant from Tolerant (SIFT; <a href="http://sift.bii.a-star.edu.sg/" target="_blank">http://sift.bii.a-star.edu.sg/</a>) and Polymorphism Phenotype (PolyPhen-2; <a href="http://genetics.bwh.harvard.edu/pph2/" target="_blank">http://genetics.bwh.harvard.edu/pph2/</a>). If the SIFT prediction tolerance index score was less than 0.05, the variation was considered possibly damaging. Predictions made by PolyPhen-2 were assigned as “probably damaging,” “possibly damaging” or “benign.” Deletion mutations cannot be tested by either SIFT or PolyPhen-2.</p

Migration analysis of EPHB6 expressing NSCLC cells.

<p>A) Protein expression of stably transfected A549 cell lines expressing wild type EPHB6 or the EPHB6 deletion mutant. Cells were co-transfected using an EGFP -pcDNA3.1⁺ vector for identification of selected clones. Multiple clones were pooled and further selected as bulk cultures. B) Transwell migration assays were performed with empty vector control cells, EPHB6 mutant and EPHB6 wildtype cells. Five different experiments in triplicates were analyzed. *: significant (p<0.05) differences by (EITHER ANOVA OR t-test) The provided p-value between the three different cell lines was statistically analyzed from all migrated cells by using the OneWay ANOVA-test. The analysis of the pair-wise t-test results in a significant p-value for the control cells vs. EPHB6-wt cells (p<0.015) and between the EPHB6-wt cells and the EPHB6-mut cells (p<0.005). C) <i>In vitro</i> wound healing scratch assay. Cells were scratched by a 10 µl pipette tip. The scratch areas were recorded over a periode of 17 hours. Shown are means of three different experiments, calculated as percentage from one initial point for all three cell lines. The ANOVA-test (p<0.002) indicated statistically significant differences between the three cell lines. D) Representative images of the scratch assays at the beginning and the end of the experiments.</p