RNAi-mediated stathmin suppression reduces lung metastasis in an orthotopic neuroblastoma mouse model

Tumour Biology and Targeting Program, Children's Cancer Institute Australia, Lowy Cancer Research Centre, University of New South Wales, Randwick, New South Wales, Australia.
Oncogene (Impact Factor: 8.46). 02/2013; 33(7). DOI: 10.1038/onc.2013.11
Source: PubMed


Metastatic neuroblastoma is an aggressive childhood cancer of neural crest origin. Stathmin, a microtubule destabilizing protein, is highly expressed in neuroblastoma although its functional role in this malignancy has not been addressed. Herein, we investigate stathmin's contribution to neuroblastoma tumor growth and metastasis. Small interfering RNA (siRNA)-mediated stathmin suppression in two independent neuroblastoma cell lines, BE(2)-C and SH-SY5Y, did not markedly influence cell proliferation, viability or anchorage-independent growth. In contrast, stathmin suppression significantly reduced cell migration and invasion in both the neuroblastoma cell lines. Stathmin suppression altered neuroblastoma cell morphology and this was associated with changes in the cytoskeleton, including increased tubulin polymer levels. Stathmin suppression also modulated phosphorylation of the actin-regulatory proteins, cofilin and myosin light chain (MLC). Treatment of stathmin-suppressed neuroblastoma cells with the ROCKI and ROCKII inhibitor, Y-27632, ablated MLC phosphorylation and returned the level of cofilin phosphorylation and cell invasion back to that of untreated control cells. ROCKII inhibition (H-1152) and siRNA suppression also reduced cofilin phosphorylation in stathmin-suppressed cells, indicating that ROCKII mediates stathmin's regulation of cofilin phosphorylation. This data demonstrates a link between stathmin and the regulation of cofilin and MLC phosphorylation via ROCK. To examine stathmin's role in neuroblastoma metastasis, stathmin short hairpin RNA (shRNA)\luciferase-expressing neuroblastoma cells were injected orthotopically into severe combined immunodeficiency-Beige mice, and tumor growth monitored by bioluminescent imaging. Stathmin suppression did not influence neuroblastoma cell engraftment or tumor growth. In contrast, stathmin suppression significantly reduced neuroblastoma lung metastases by 71% (P<0.008) compared with control. This is the first study to confirm a role for stathmin in hematogenous spread using a clinically relevant orthotopic cancer model, and has identified stathmin as an important contributor of cell invasion and metastasis in neuroblastoma.Oncogene advance online publication, 11 February 2013; doi:10.1038/onc.2013.11.

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Available from: Frances Byrne, Sep 05, 2014
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    • "We observed a significant reduction in tumour cell proliferation and cell invasiveness , in vitro , when decreasing the STMN1 protein levels by using siRNA - targeting STMN1 . This is in agreement with other in vitro and in vivo studies , where inhibition of STMN1 reduced tumour growth and cell invasion ( Baldassarre et al , 2005 ; Hsieh et al , 2010 ; Jeon et al , 2010 ; Zheng et al , 2010 ; Lei et al , 2011 ; Phadke et al , 2011 ; Tan et al , 2012 ; Byrne et al , 2013 ; Chen et al , 2013 ) . Our results indicate that STMN1 is of importance for tumour cell proliferation and migration also in bladder cancer . "
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    ABSTRACT: Background: The oncoprotein-18/stathmin 1 (STMN1), involved in cell progression and migration, is associated with clinical outcome in breast cancer. Here we aim to investigate its clinical significance in urinary bladder cancer and its possibilities as a therapeutic target. Methods: Immunohistochemical analyses of STMN1 protein expression were performed in three patient cohorts: cohort I (n=115 Ta, n=115 T1, n=112 T2–4 stages), cohort II, based on randomised controlled trials (n=239 T1–T4), and cohort III of primary tumour/matched metastasis (n=90 T1–T4). The effects of STMN1 on cell proliferation and migration were evaluated in the urinary bladder cancer cell line, T24, by inhibiting STMN1-cellular expression using siRNA. Results: In cohort I, high STMN1 expression correlated to shorter disease-specific survival hazard ratio (HR)=2.04 (95% confidence interval (CI) 1.13–3.68; P=0.02), elevated p53- (P<0.001) and Ki67-protein levels (P<0.001). The survival result was validated in cohort II: HR=1.76 (95% CI 1.04–2.99; P=0.03). In the metastatic bladder cancer material, 70% of the patients were STMN1-positive in both the primary tumour and matched metastases. In vitro, the growth and migration of the T24 cells were significantly reduced (P<0.01, P<0.0001, respectively), when transfecting the cells with STMN1-siRNA. Conclusions: STMN1 protein expression has prognostic significance but is primarily a potential treatment target in urinary bladder cancer.
    British Journal of Cancer 09/2014; 111(7):1180-1187. DOI:10.1038/bjc.2014.427 · 4.84 Impact Factor
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    • "These steps require a high motility of cancer cells, which is mediated by modulation of the cellular cytoskeleton (for review see [2,3]). Recent studies indicate cytoskeleton binding proteins as important players in tumor metastasis [4,5] (for review see [6]), particularly by their capability to bind to and regulate integrin molecules [7]. Therefore, such molecules may be promising targets to inhibit the metastatic properties of tumor cells. "
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    ABSTRACT: Tumor cell migration and metastasis require dynamic rearrangements of the actin cytoskeleton. Interestingly, the F-actin cross-linking and stabilizing protein L-plastin, originally described as a leukocyte specific protein, is aberrantly expressed in several non-hematopoietic malignant tumors. Therefore, it has been discussed as a tumor marker. However, systematic in vivo analyses of the functional relevance of L-plastin for tumor cell metastasis were so far lacking. We investigated the relevance of L-plastin expression and phosphorylation by ectopical expression of L-plastin in human melanoma cells (MV3) and knock-down of endogenous L-plastin in prostate cancer (PC3M). The growth and metastatic potential of tumor cells expressing no L-plastin, phosphorylatable or non-phosphorylatable L-plastin was analyzed in a preclinical mouse model after subcutaneous and intracardial injection of the tumor cells. Knock-down of endogenous L-plastin in human prostate carcinoma cells led to reduced tumor cell growth and metastasis. Vice versa, and in line with these findings, ectopic expression of L-plastin in L-plastin negative melanoma cells significantly increased the number of metastases. Strikingly, the metastasis promoting effect of L-plastin was not observed if a non-phosphorylatable L-plastin mutant was expressed. Our data provide the first in vivo evidence that expression of L-plastin promotes tumor metastasis and, importantly, that this effect depends on an additionally required phosphorylation of L-plastin. In conclusion, these findings imply that for determining the importance of tumor-associated proteins like L-plastin a characterization of posttranslational modifications is indispensable.
    Molecular Cancer 01/2014; 13(1):10. DOI:10.1186/1476-4598-13-10 · 4.26 Impact Factor
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    ABSTRACT: Dynamic microtubule changes following a cell stress challenge are required for cell survival and adaptation. Stathmin (STMN), a cytoplasmic microtubule-destabilizing phosphoprotein, regulates interphase microtubules during cell stress but the signalling mechanisms involved are poorly defined. In this study, through the ectopic expression of single alanine-substituted phospho-resistant mutants demonstrated that STMN S38 and S63 phosphorylation were specifically required to maintain interphase microtubules during hyperosmotic stress (OS). STMN was phosphorylated on S38 and S63 in response to hyperosmolarity, heat shock and arsenite treatment but rapidly dephosphorylated following oxidative stress treatment. 2D-PAGE and Phos-tag gel analysis of stress-stimulated STMN phospho-isoforms revealed rapid STMN S38 phosphorylation followed by subsequent S25 and S63 phosphorylation. Previously, we delineated stress-stimulated JNK targeting of STMN. Here, we identified cAMP-dependent protein kinase (PKA) signalling as responsible for stress-induced STMN S63 phosphorylation. Increased cAMP levels induced by cholera toxin triggered potent STMN S63 phosphorylation. OS stimulated an increase in PKA activity and elevated STMN S63 and CREB S133 phosphorylation that was substantially attenuated by pretreatment with H-89, a PKA inhibitor. Interestingly, PKA activity and subsequent phosphorylation of STMN were augmented in absence of JNK activation indicating JNK and PKA pathway cross-talk during stress regulation of STMN. Taken together our study indicates that JNK and PKA-mediated STMN S38 and S63 phosphorylation are required to preserve interphase microtubules in response to hyperosmotic stress.
    Journal of Biological Chemistry 12/2013; 289(4). DOI:10.1074/jbc.M113.470682 · 4.57 Impact Factor
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