JNK1 determines the oncogenic or tumor-suppressive activity of the integrin-linked kinase in human rhabdomyosarcoma
Adam D. Durbin, Gino R. Somers, Michael Forrester, Malgorzata Pienkowska, Gregory E. Hannigan, David Malkin
Adam D. Durbin, Gino R. Somers, Michael Forrester, Malgorzata Pienkowska, Gregory E. Hannigan, David Malkin
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Research Article

JNK1 determines the oncogenic or tumor-suppressive activity of the integrin-linked kinase in human rhabdomyosarcoma

Abstract

Although most reports describe the protein kinase integrin-linked kinase (ILK) as a proto-oncogene, occasional studies detail opposing functions in the regulation of normal and transformed cell proliferation, differentiation, and apoptosis. Here, we demonstrated that ILK functions as an oncogene in the highly aggressive pediatric sarcoma alveolar rhabdomyosarcoma (ARMS) and as a tumor suppressor in the related embryonal rhabdomyosarcoma (ERMS). These opposing functions hinge on signaling through a noncanonical ILK target, JNK1, to the proto-oncogene c-Jun. RNAi-mediated depletion of ILK induced activation of JNK and its target, c-Jun, resulting in growth of ERMS cells, whereas in ARMS cells, it led to loss of JNK/c-Jun signaling and suppression of growth both in vitro and in vivo. Ectopic expression of the fusion gene characteristic of ARMS (paired box 3–forkhead homolog in rhabdomyosarcoma [PAX3-FKHR]) in ERMS cells was sufficient to convert them to an ARMS signaling phenotype and render ILK activity oncogenic. Furthermore, restoration of JNK1 in ARMS reestablished a tumor-suppressive function for ILK. These findings indicate what we believe to be a novel effector pathway regulated by ILK, provide a mechanism for interconversion of oncogenic and tumor-suppressor functions of a single regulatory protein based on the genetic background of the tumor cells, and suggest a rationale for tailored therapy of rhabdomyosarcoma based on the different activities of ILK.

Authors

Adam D. Durbin, Gino R. Somers, Michael Forrester, Malgorzata Pienkowska, Gregory E. Hannigan, David Malkin

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Figure 5

Loss of ILK induces opposing effects on the JNK/c-Jun signaling axis sensitive to PAX3-FKHR in vitro and in vivo.

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Loss of ILK induces opposing effects on the JNK/c-Jun signaling axis sen...
(A) RD and Rh6 (ERMS) as well as Rh4 and Rh18 (ARMS) cell lines were transfected with nothing or with ILK or control siRNAs and lysed for Western blotting. Arrows show p54 and p46 JNK proteins. Blots and densitometry represent the average of 4 independent experiments. (B) RMS xenografts were lysed for Western blotting. Data are representative of 6 tumors per treatment, per cell line. Arrows show p54 and p46 JNK proteins. (C) RD (ERMS) and Rh4 (ARMS) cells were transfected with ILK or control siRNAs and lysed for RNA. Quantitative RT-PCR for c-Jun/AP-1–regulated transcripts was performed (n = 4). Values denote transcript fold change relative to control siRNA treatment. All differences were significant (P < 0.001) compared with internal control siRNA. (D) RD vector and PAX3-FKHR clones were transfected with nothing or with ILK or control siRNAs. Lysates were prepared for Western blotting. Data are representative of 3 independent blots and normalized against lipofectamine-treated RD/pcDNA-1 cells. Arrows denote p46 and p54 JNK proteins. Phospho-JNK lanes were run on the same gel but were noncontiguous (white line). (E) ARMS primary tumors were divided into low (0–5; n = 22) and high (6–9; n = 13) ILK score groups, and phospho-JNK score was determined. ###P = 0.0031 versus low ILK score. (F) ERMS primary tumors were divided into low (0–6; n = 17) and high (7–9; n = 19) ILK score groups, and phospho-JNK score was determined. *P = 0.038 versus low ILK score. (G) ERMS samples were divided by tumor stage, and phospho-JNK score was determined (n = 10 [I]; 12 [II]; 11 [III/IV]). **P < 0.01 versus stage I and stage II, ANOVA.