Interplay between microRNA-21 and Sprouty2 in non small cell lung cancer-derived cell lines

Einer einzelnen microRNA ist es moglich uber die Bindung an den 3’ untranslatierten Bereich hunderte mRNAs zu regulieren. Von einigen microRNAs ist bekannt, dass sie in Krebs dereguliert sind. Zu dieser Gruppe von microRNAs gehört auch microRNA-21; sie agiert als sogenanntes oncomiR und ist in einer Vielzahl verschiedener Tumoren überexpremiert. Bekannte Substrate von miR-21 sind Tumorsupressoren wie ProgrammedCellDeath4, PhosphataseAndTensinHomolog, Sprouty1 und Sprouty2. In dieser Studie haben wir die microRNA-21 Expression im Lungenkrebs untersucht und herausgefunden, dass in kultivierten Lungenkrebszelllinien die Levels von microRNA-21 kaum von normalen Lungenfibroblasten abweichen. Die Korrelation microRNA-21 Expression und Proteinlevel von ProgrammedCellDeath4, PhosphataseAndTensinHomolog und Sprouty war insignifikant. Die Proliferationsrate der Zelllinien stand in keinem Zusammenhang zu den gemessenen miR-21 Levels ebenso wie der Unterschied von microRNA-21 Expression in Adenokarzinomen und Plattenzellkarzinomen. Zelllinien, die eine onkogene Mutation im Ras Gen habenzeigen im Mittel zwar geringfügig höhere microRNA-21 Werte, aber eine ektopische Expression von onkogen mutierten H-, K- und N-Ras führte zu keiner Veränderung der microRNA-21 Expression Während Überexpression von Sprouty1 keinen Effekt auf microRNA-21 hat, führen hohe Levels von Sprouty2 zu einer signifikanten Reduktion der microRNA-21 Expression. Zudem wurden verschiedene Strategien zur Modulierung der microRNA-21 Expression verglichen. Überexpression von verkürzten primiR 21 RNA durch ein virales System zeigte die gewünschte Wirkung. Um microRNA-21 zu inhibieren wurden verschiedene Ansatze ausprobiert, wobei nur Zellen der Zelllinie U373, die mit dem sponge eraser Konstrukt infiziert wurden, signifikant reduzierte microRNA-21 Expression zeigen. Diese Daten fuhren zu der Annahme, dass die Produktion von sponge eraser Konstrukten die beste Strategie ist.MicroRNAs can regulate hundreds of mRNA by binding at the 3’ untranslatedRegions. Some microRNAs are known to be deregulated in cancer. MicroRNA-21is known to act as an oncomiR. Identified targets of miR-21 are tumor suppressors like ProgrammedCellDeath4, PhosphataseAndTensinHomolog,Sprouty1 and Sprouty2. In this study we investigated miR-21 expression in non small cell lung cancer (NSCLC) derived cell lines and discovered that in cultured NSCLC derived cell lines microRNA-21 expression is comparable to the levels in normal lungfibroblasts. MicroRNA-21 levels failed to show inverse correlation to the protein expression of ProgrammedCellDeath4, PhosphataseAndTensinHomolog and Sprouty. We were unable to detect correlation between expression of miR-21and proliferation rate as well no correlation of microRNA-21 expression between cell lines derived from adenocarcinoma and squamous cell carcinoma was found. Although cell lines harboring an oncogenic mutation in the Ras gene show on average higher levels, overexpression of oncogenic H-, K- and N-Ras does not lead to an alteration of microRNA-21 expression. While overexpression of Spry1 has no effect on miR-21 levels, high levels of Spry2 lead to reduced microRNA-21 expression. Additionally we compared different strategies to modulated miR-21levels. Overexpressing of a shortened primiR 21 RNA worked at least in one of the chosen cell lines. In order to achieve inhibition of microRNA-21 we tested different strategies. Only cells from U373 cell lines, which were infected with a so called sponge eraser construct show significant reduced microRNA-21 levels. These data indicate that production of sponge eraser constructs is the most powerful way to interfere with microRNA-21

Sprouty2 and Sprouty4 exert distinguishable tumor-repressing effects in osteosarcoma and breast cancer, but only Sprouty2 is able to rescue the oncogenic functions of microRNA-21

Sprouty Proteine spielen eine wichtige Rolle in der Regulation von Signaltransduktion. Über unterschiedliche Mechanismen ist es ihnen meistens möglich, Signalkaskaden abzuschwächen. Nur in wenigen Fällen -wohl weitgehend abhängig vom extern vorhandenen Signalinduktor kann ein erhöhter Level an Sprouty Proteinen die Signalwirkung in der Zelle auch verstärken. Dementsprechend ist auch die ihnen nachgewiesene Rolle bei der Tumorentstehung höchst unterschiedlich. Da einzelne Funktionen, insbesondere von Sprouty2 und Sprouty4 unterschiedlich ausgeübt werden können, haben wir beschlossen, die Wirkungsweise dieser beiden Sprouty Proteine in Brustkrebs und Osteosarkom-Zelllinien vergleichend zu untersuchen. In Brustkrebszellen war Sprouty4 effizienter bei der Inhibierung der Zellproliferation und der Migration als Sprouty2. Dazu passend konnte gezeigt werden, dass in der Brustkrebs-Zelllinie mit erhöhter Sprouty4 Expression eine Reduktion der Sprouty4 Proteinlevels zu einer gesteigerten Proliferation geführt hat. Die stärker inhibierende Wirkung von Sprouty4 ist vermutlich durch einen verminderten Anstieg der Phosphorylierung von ERK in Folge von Seruminduktion verursacht. Ganz im Gegensatz zu diesen Daten, war in Osteosarkomzellen, ausschließlich Sprouty2 in der Lage eine tumorsuppressive Funktion zu erfüllen. Bei ektopischer Sprouty2 Überexpression stellen Osteosarkom-Zelllinien die Zellteilung nahezu ein. Interessanterweise hemmt in diesem Zelltyp Sprouty2 die Aktivierung des MAPK Signaltransduktionsweges wesentlich effizienter als Sprouty4. N-terminal gelegene Strukturen sind für diesen Unterschied verantwortlich. Obwohl die signifikante Repression von Sprouty2 Expression in Osteosarkomen noch nicht eindeutig untersucht wurde, ist bekannt, dass ein wichtiger Regulator von Sprouty2, nämlich mikroRNA-21 in Osteosarkomen erhöht ist. In meiner Thesis konnte ich zeigen, dass Osteosarkomzellen mit erhöhten mikroRNA-21 Levels durch eine schnellere Zellverdoppelung charakterisiert sind. Umgekehrt verlangsamt sich die Proliferation von Osteosarkomzellen, wenn die Aktivität von mikroRNA-21 reduziert wird. Zusätzlich zeigte sich, dass die endogenen Sprouty2 Levels auf die Modulation der mikroRNA-21 Aktivitäten reagieren, und dass die ektopische Expression von Sprouty2 die Wirkung von mikroRNA-21 Überexpression komplett aufhebt. Neben der proliferationsfördernden Wirkung konnte mikroRNA-21 auch die Sensitivität der Osteosarkomzellen gegenüber Cisplatin erhöhen, während Migration und die Empfindlichkeit der Zellen gegenüber Doxorubicin und Methotrexat von den mikroRNA-21 Expressionslevel unbeeinflusst bleiben. Die erhöhte Sensitivität gegenüber Cisplatin im Falle von erhöhten mikroRNA-21 Levels ist bei gleichzeitiger Überexpression von Sprouty2 teilweise aufgehoben. Diese Ergebnisse zeigen deutlich, dass die Regulation von Sprouty2 durch mikroRNA-21 zumindest im Osteosarkom bedeutend ist. In der Lunge, wo Sprouty2 auch als Tumorsuppressor wirkt, ist mikroRNA-21 zwar im Tumorgewebe deutlich höher als im dazugehörigen Normalgewebe, diese Erhöhung kann aber in Zelllinien nur beobachtet werden, wenn tumorspezifische Wachstumsbedingungen wie Hypoxie oder Sphäroides Zellwachstum simuliert werden. Zusammenfassend können wir sagen, dass die Sensitivität des MAPK Signaltransduktionsweg auf Spouty2 und Sprouty4 in Brustkrebszellen und Osteosarkomzellen unterschiedlich ist. Damit einhergehend ist das Ausmaß der hemmenden Wirkung der beiden Sprouty Proteine auf Wachstum und Migration klar zu unterscheiden. Die Regulation von Sprouty2 durch das OnkomiR mikroRNA-21 dürfte in Osteosarkomen entscheidend sein.Sprouty proteins are important modulators of receptor tyrosine kinase-mediated signaling. Primarily, these proteins suppress signaling cascades like MAPK pathway. Nonetheless, there is evidence that in response to certain extracellular signal conditions at least Sprouty2 is able to sustain cell signaling. Due to these opposing roles in signal transduction, Sprouty proteins can function as tumor suppressors as well as oncogenes, mainly dependent on the origin of the cancer. Since the available data indicate that especially Sprouty2 and Sprouty4 may exert distinguishable roles, we first compared the function of these two Sprouty proteins in breast cancer- as well as osteosarcoma-derived cells. In breast cancer-derived cell lines, we observed that compared to Sprouty2, Sprouty4 is the more potent inhibitor of cell proliferation and migration. Accordingly, knock-down of Sprouty4 levels in cells with high endogenous expression of Sprouty4 decelerated cell proliferation. Concurrently, the induction of ERK in response to serum in the tested breast cancer cells was primarily repressed by Sprouty4 indicating that the impact of Sprouty4 on cell proliferation was due to interference with the MAPK pathway. In contrast to the observations in breast cancer, we demonstrated that Sprouty2 acts as a potent tumor suppressor in osteosarcoma, where Sprouty4 fails to influence malignant characteristics of the cells. Ectopic expression of Sprouty2 almost arrested cell proliferation in osteosarcoma-derived cells. Both Sprouty proteins are able to interfere with MAPK pathway activation, but Sprouty2 inhibits ERK phosphorylation much more efficient. The difference in this function arises from structures located at the N-terminus of Sprouty2. Although, Sprouty2 expression in osteosarcoma tissue is not documented yet, it is reported that microRNA-21, an important regulator of Sprouty2, is frequently overexpressed. We modulated microRNA-21 activity, either by expressing a sponge construct for suppressing its activity or by overexpression of primiR-21. Our study illustrates that microRNA-21 accelerated cell proliferation and ectopic expression of Sprouty2 in microRNA-21 overexpressing cells compensated this effect. Contrarily, modulation of microRNA-21 left cell migration unaffected. In addition, augmented microRNA-21 activity led to increased sensitivity of the cells towards cisplatin treatment, while it had no effect on resistance to other commonly used chemotherapeutic agents like doxorubicin and methotrexate. Simultaneous ectopic expression of Sprouty2 and microRNA-21 reversed the microRNA-21-mediated effect on cell sensibility towards cisplatin treatment. Our data suggest that the regulation of Sprouty2 by microRNA-21 plays an important role in osteosarcoma. In lung cancer, where Sprouty2 is reported to act as a tumor suppressor, microRNA-21 is clearly elevated in tumor tissue compared to the corresponding normal tissue. Surprisingly, this increase in microRNA-21 expression could only be observed in NSCLC-derived cell lines after simulation of tumor-specific growth conditions like hypoxia or anchorage-independent growth. In summary, we concluded that the influence of Sprouty2 and Sprouty4 on MAPK signaling cascade differs in breast cancer and osteosarcoma. In line with this observation, the effect of the two Sprouty isoforms on migration and proliferation varies clearly in these two tumor entities. Additionally, the regulation of Sprouty2 by microRNA-21 seems to be essential for the malignant phenotype of osteosarcoma.submitted by Mag. rer. nat. Vanita VanasZusammenfassung in deutscher SpracheMedizinische Universität Wien, Dissertation, 2016OeBB(VLID)192060

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

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

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 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

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 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 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