Tumor-associated growth conditions activate mechanisms regulating Sprouty protein expression

Mitogen-aktivierte Proteinkinase (MAPK) Signalwege stellen essentielle Signaltransduktionskaskaden dar, welche eine Vielzahl zellulärer Prozesse wie etwa Proliferation, Differenzierung, Apoptose und Migration beeinflussen. Diese Signalwege werden durch verschiedene Mechanismen moduliert, wie z.B. durch die Regulationsproteine Sprouty1-4 (Spry1-4). Die Mitglieder der Spry Proteinfamilie besitzen überlappende Wirkungsmechanismen, jedoch sind ihre Funktionen nicht redundant. Spry Proteine üben eine zentrale Rolle in der proliferationsregulierenden Signaltransduktion aus. Häufig ist die Expression von Spry Proteinen, vor allem Spry2, in verschiedenen Tumorarten wie etwa dem Lungenkarzinom vermindert. Daher wird angenommen dass Spry Proteine eine tumorsupprimierende Funktion ausüben. Basierend auf den kürzlich publizierten Daten dass Spry2 durch microRNA-21 (miR-21) reguliert wird, untersuchten wir eine potenzielle Beeinflussung von Spry2 durch miR-21 im Lungenkrebs. Daher analysierten wir im Zuge dieser Dissertation die miR-21 Mengen in Zelllinien und Geweben des nicht-kleinzelligen Lungenkarzinoms. Übereinstimmend mit früheren Studien weisen unsere Resultate nach dass miR-21 verstärkt im Tumor vorhanden ist verglichen mit Normalgewebe. Zusätzlich konnten wir zeigen dass der Anstieg von miR-21 mit zunehmender Malignität ausgeprägter ist. Wie zu erwarten war wiesen jene Tumore mit besonders hoher miR-21 Menge eine stark reduzierte Spry2 Expression auf. Im Gegensatz zur Erhöhung von miR-21 in Geweben waren die miR-21 Mengen in den Zelllinien vergleichbar mit denen im Normalgewebe und nicht-malignen Zellen. Dementsprechend fanden wir keine Korrelation zwischen den Levels an miR-21 und der Spry2 Expression in Zelllinien des nicht-kleinzelligen Lungenkarzinoms. Trotzdem war die Beobachtung dass miR-21 ausschließlich im Gewebekontext erhöht wurde besonders interessant weil diese Daten darauf hinwiesen dass Komponenten des Tumormikromilieus, die nicht in der Zellkultur nachgeahmt werden, eine entscheidende Rolle in der Regulierung von miR-21 spielen. Deshalb untersuchten wir den Einfluss von Tumor-assoziierten Bedingungen auf miR-21. Während Tumor-assoziierte Fibroblasten die Expression von miR-21 nicht beeinflussten, führten sowohl kontaktunabhängiges Wachstum als auch Hypoxie zu einem Anstieg der miR-21 Menge in Zelllinien des nicht-kleinzelligen Lungenkarzinoms. Diese Ergebnisse zeigen dass miR-21 vorwiegend durch gewebespezifische und nicht intrinsische Faktoren reguliert wird. In Zellen des nicht-kleinzelligen Lungenkarzinoms, die kontaktunabhängig wuchsen oder unter Hypoxie kultiviert wurden, waren die Level an Spry2 vermindert. Diese Resultate lassen eine Regulierung von Spry2 durch miR-21 vermuten. Im Gegensatz zu den Daten die für Spry2 erhalten wurden, zeigten die Ergebnisse der Spry4 Expressionsanalysen in verschiedenen Zelllinien dass Spry4 unter hypoxischen Bedingungen hochreguliert wird. Der Anstieg der Spry4 Expression in Folge von Eisenmangel war unabhängig von Zelltyp, Differenzierung und Bösartigkeit. Des weiteren deckte unsere Studie eine Mitogen-unabhängige Regulation von Spry4 auf. Eine genauere Untersuchung der zugrundeliegenden Mechanismen ergab dass hauptsächlich erhöhte mRNA Stabilität und Transkriptionsaktivität für den Anstieg von Spry4 bei Eisenentzug verantwortlich sind, wohingegen Spry4 Proteine destabilisiert werden. Eine Mitwirkung des zentralen Transkriptionsfaktors HIF-1 (Hypoxie-induzierter Faktor-1) an der Spry4 Promoterregulation konnte gezeigt werden. Die Analyse der MAPK Signaltransduktion unter hypoxischen Bedingungen deutete eine Hemmung dieses Signalweges durch Spry4 an. Unsere Resultate indizieren eine Beteiligung von Spry4 an der zellulären Adaption an Sauerstoffmangel. Zusammenfassend konnten die Daten der Dissertation aufzeigen dass die Expression der Spry Proteine durch unterschiedliche Regulationsmechanismen kontrolliert wird um eine adäquate Verfügbarkeit der verschiedenen Mitglieder der Spry Proteinfamilie zu gewährleisten.Mitogen-activated protein kinase (MAPK) pathways represent key signalling cascades that interfere with a broad range of cellular processes such as proliferation, differentiation, apoptosis and migration. These pathways are modulated by various mechanisms, including the negative-feedback regulators Sprouty1-4 (Spry1-4). Spry family members are known to have overlapping modes of action but their functions are not redundant. Spry proteins fulfill a central role in proliferative signalling and are frequently downregulated in diverse malignancies. Hence they are assumed to be tumor suppressors. Especially Spry2 expression is suppressed in many tumor entities including lung cancer. Since microRNA-21 (miR-21) was recently reported to target Spry2, we investigated the potential impact of miR-21 on Spry2 expression in lung cancer. In this thesis we analysed miR-21 levels in Non-small cell lung cancer (NSCLC)-derived tissue samples and cell lines. Consistent with earlier studies, the obtained results demonstrate that miR-21 is elevated in tumor versus normal lung tissue. Additionally we unveiled that with progression of the disease the increase of miR-21 is considerably more pronounced. As expected, lung tumor tissues with high miR-21 levels tended to express low Spry2 amounts. In contrast to the lung tumor tissues, NSCLC-derived cell lines exhibited miR-21 levels comparable to those of non-malignant tissues and cell lines. Accordingly, we found no correlation between miR-21 levels and Spry2 expression in NSCLC-derived cell lines. Nonetheless the observation that miR-21 was upregulated solely in the tissue context was particularly interesting since the data indicated that microenvironmental conditions, which are not mimicked in cell culture, play an important role in regulation of miR-21. Hence we studied the influence of tumor-associated environmental factors on miR-21. While the expression of miR-21 was not affected by cancer-associated fibroblasts, both oxygen withdrawal and anchorage-independent growth led to augmented miR-21 levels in NSCLC-derived cell lines. These results highlight that miR-21 is regulated by tissue-specific microenvironmental conditions rather than intrinsic factors. NSCLC-derived cells growing anchorage-independently or under hypoxia harboured reduced Spry2 levels, pointing to miR-21-mediated regulation of Spry2. In contrast to the data obtained for Spry2, evaluation of Spry4 expression under hypoxic conditions revealed that Spry4 is upregulated in diverse cell lines. The increase of Spry4 expression following iron deprivation was irrespective of cell origin, differentiation and malignancy. Moreover, this study uncovered a mitogen-independent regulation of Spry4. A detailed analysis of the underlying mechanisms showed that primarily enhanced mRNA stability and augmented transcriptional activity account for Spry4 enhancement, while Spry4 protein was destabilized upon iron withdrawal. Hypoxia-inducible factor-1, a main transcriptional regulator during hypoxia, was shown to be involved in Spry4 promoter control. Analysis of MAPK signalling illustrated that Spry4 presumably suppresses this pathway under hypoxic conditions, indicating that Spry4 contributes to the cellular adaption to hypoxia. In summary our data demonstrate that the expression of Spry proteins is controlled by distinct regulatory mechanisms in order to ensure appropriate availability of the different Spry protein family members.Abweichender Titel laut Übersetzung der Verfasserin/des VerfassersWien, Med. Univ., Diss., 2013OeBB(VLID)171365

MicroRNA-21 Increases Proliferation and Cisplatin Sensitivity of Osteosarcoma-Derived Cells.

Osteosarcoma is the most common primary bone tumor and poor prognosis for osteosarcoma patients is mainly due to chemotherapy resistance. MicroRNAs are important to maintain pathophysiological mechanisms of cancer and influence cell sensitivity to chemotherapy. In this study, we tested the functions of microRNA-21 for malignant features as well as for drug resistance of osteosarcoma. We used Northern blot to measure microRNA-21 levels in osteosarcoma-derived cell lines. MicroRNA-21 activity was modulated by either expressing a sponge to decrease its activity in an osteosarcoma-derived cell line expressing high levels of microRNA-21 or by introducing pri-microRNA-21 in a cell line with low endogenous levels. Cell migration was determined in a scratch assay and cell proliferation was measured by performing growth curve analysis. Sensitivity of the cells towards chemotherapeutics was investigated by performing cell viability assays and calculating the IC50 values. While cell migration was unaffected by modulated microRNA-21 levels, microRNA-21 inhibition slowed proliferation and exogenously expressed microRNA-21 promoted this process. Modulated microRNA-21 activity failed to effect sensitivity of osteosarcoma-derived cell lines to doxorubicin or methotrexate. Contrarily, reduction of microRNA-21 activity resulted in enhanced resistance towards cisplatin while ectopic expression of microRNA-21 showed the opposite effect. Increased microRNA-21 levels repressed the expression of Sprouty2 and ectopic expression of Sprouty2 was able to largely rescue the observed effects of microRNA-21 in osteosarcoma. In summary, our data indicate that in osteosarcoma microRNA-21 expression is an important component for regulation of cell proliferation and for determining sensitivity to cisplatin

Influence of reduced miR-21 activity on cell proliferation and migration.

<p>U2OS were transfected with either pBabepuro (pBp), pBabepuro luciferase (pBluc) or a miR-21 sponge. (A) Cell proliferation was measured by counting the cells daily. A representative growth curve is depicted (Means ± SEM). Using exponential growth equation, doubling times were calculated using Graph Pad Prism 5 (Means ± SEM). Values from six experiments of three clones are depicted and significance was calculated. **p < 0.01 (B) A scratch assay with U2OS cells transfected with the indicated plasmids was performed. Representative pictures of cells 1 and 12 hours after scratch was set are shown (upper panel). Migration velocity was calculated using linear regression. Means ± SEM of three independent experiments are shown (lower panel). (C) Transfection efficiency was verified by measuring luciferase activity of the selected mixed clones (MC1-3) (Means ± SEM).</p

Influence of Spry2 and Spry4 expression on proliferation of miR-21 overexpressing cells.

<p>Prior to growth curve analysis, MG63 empty vector or primiR-21 expressing clones were infected with either an adenovirus expressing lacZ (control), Spry2 or Spry4. (A) Representative curves depicting means ± SEM are presented. The ectopically expressed genes are indicated as legend. (B) Doubling times of three to six independent experiments were calculated and depicted as means ± SEM. Significance was determined by unpaired t-test. *p < 0.05; **p < 0.01; ***p < 0.001 (C) Protein lysates of the infected clones were analyzed by performing an immunoblot with the indicated antibodies.</p

Influence of reduced miR-21 activity on the sensitivity of U2OS cells towards chemotherapeutic drugs.

<p>Logarithmically growing MCs were incubated for 48 hours to the indicated increasing concentration of cisplatin (A), methotrexate (B) or doxorubicin (C) and a cell viability assay was performed (left panel). Means ± SEM of a representative experiment are presented as curves. The IC50 values of three to four experiments are depicted as means ± SEM and an unpaired t-test was performed; *p < 0.05 (right panel).</p

Influence of ectopic miR-21 expression on cell proliferation and cisplatin sensitivity of MG63 cells.

<p>Cells were transfected with pBp or primiR-21 and single clones were selected. (A) A representative growth curve of one clone is depicted as means ± SEM. Doubling times of six experiments with three different clones were calculated, the means ± SEM are presented in a column graph and analyzed using unpaired t-test. **p < 0.01. (B) A cell viability assay of a representative single clone obtained after transfection with the indicated plasmid is presented as means ± SEM. IC50 values of three different clones were calculated. Means ± SEM summarize the data. The significance was determined using an unpaired t-test. **p < 0.01 (C) MiRNA Northern blots of the indicated clones obtained after transfection with primiR-21or pBp are shown.</p

Influence of ectopic Spry protein expression on cisplatin-sensitivity of MG63 cells with different miR-21 expression levels.

<p>MG63 clones expressing an empty vector (pBp) or a primiR-21 were infected with adenoviruses expressing the indicated proteins. LacZ was used as control protein. (A) The cell viability assays of representative clones infected with the indicated proteins (see legend) are shown as curves (Means ± SEM). (B) IC50 values of three different clones are summarized as means ± SEM and a t-test was performed. *p < 0.05.</p

Influence of ectopic miR-21 expression on potential target proteins.

<p>Protein lysates of cell clones obtained after transfection and selection of MG63 cells with either pBp or primiR-21 were analyzed using an immunoblot. Protein expression was calculated using Image quant 5.0 software. The highest value was arbitrarily set as 1. GAPDH was used as reference value. (A) Means ± SEM of two to three independent experiments are depicted. The analyzed protein is indicated as title. (B) Calculated levels of the indicated proteins in the clones overexpressing miR-21 were compared to the one in the corresponding control clones. Means ± SEM are presented. Significance was calculated using unpaired t-test. *p < 0.05; **p < 0.01.</p

Endogenous expression of miR-21 in osteosarcoma-derived cell lines.

<p>Logarithmically growing cells were harvested to perform a miRNA Northern blot. (A) One representative Northern blot measuring miR-21 levels of six osteosarcoma-derived cell lines is shown. U6 served as loading control. (B) Results of two to three Northern blots were quantified using Image Quant 5.0. The miR-21/U6 ratios were determined and depicted as a block diagram. Means ± SEM are shown.</p