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The cytoplasmic truncated receptor tyrosine kinase ALK homodimer immortalizes and cooperates with ras in cellular transformation^{1 ,2}

INGRID SIMONITSCH^*,3, DORIS POLGAR^1,3, MARIA HAJEK, PETER DUCHEK^*, BARBARA SKRZYPEK, SANDRA FASSL^*, ANDREA LAMPRECHT^*, GERLINDE SCHMIDT, GEORG KRUPITZA^* and CHRISTA CERNI⁴

^* Institute of Clinical Pathology,
Institute of Cancer Research, University of Vienna, A-1090 Vienna, Austria

⁴Correspondence: Institute of Cancer Research, University of Vienna, Borschkegasse 8a, A-1090 Vienna, Austria. E-mail: Christa.Cerni{at}.univie.ac.at

SPECIFIC AIM

The chromosomal t(2;5) translocation is characteristic for a subgroup of non-Hodgkin’s lymphomas, the so-called anaplastic large cell lymphomas (ALCL), which account for 1000 new cases per year in the U.S. This specific aberration results in the fusion of truncated anaplastic lymphoma kinase (ALK), a developmentally important receptor tyrosine kinase, to the oligomerization domain of nucleophosmin (NPM), a ubiquitous cellular shuttle protein. The objective of this study was to molecularly dissect the oncogenicity of the chimeric NPM/ALK protein in vitro and in vivo.

Principal findings

Malignant transformation of mammalian cells in vitro as well as in vivo requires the combined action of two classes of oncogenes, collectively termed immortalizing and transforming oncogenes, which override G1 cell cycle arrest mechanisms and activate crucial cellular signaling pathways, respectively. Thus, we investigated the contribution of NPM/ALK to tumorigenicity in primary rat embryonic cells (REC) with emphasis on its potential to cooperate with other oncogenes.

1. NPM/ALK immortalizes REC
Based on its effects on established rodent cell lines, it was suggested that NPM/ALK might be a transforming oncogene. Therefore, we tested the cooperation of NPM/ALK with the immortalizing oncogene c-myc toward cellular transformation and compared the assumed cooperative action of this oncogene combination with the well-established and efficient c-myc/c-H-ras transformation in primary REC. After having passed 20 population doublings (PDL), clones developing from heterogenous primary REC cultures can be considered immortal. Figure 1 shows growth curves of a comparative immortalization experiment. Of 14 isolated myc-only clones, 8 grew readily, 2 with delay, and 4 senesced (Fig. 1a ). C-myc and the additional presence of c-H-ras yielded transformed clones, which proliferated rapidly after isolation (Fig. 1b ). Unexpectedly, 6 of the 14 clones isolated from NPM/ALK/neo-transfected cultures also continued growing and passed the critical replicative senescence point similar to the majority of c-myc-clones (Fig. 1c ). Cotransfection of c-myc and NPM/ALK showed no cooperative effect of the two oncogenic DNAs with regard to either cell morphology, efficiency of establishment, or growth kinetics of the individual cell lines (Fig. 1d ). In primary REC of gestation day 15.5, the frequency of spontaneous immortalization is low, and only very occasionally do clones from neo- or c-H-ras-transfected DNAs pass 20 PDL after 3 months and more. Accordingly, the colonies from the vector/neo and vector/c-H-ras cultures, which were of comparable clonal size at the time of isolation, ceased to grow and senesced (Fig. 1e , f ). From these data we concluded that NPM/ALK does not cooperate with c-myc, but instead has a definite intrinsic immortalizing capacity.

View larger version (36K): [in this window] [in a new window]   Figure 1. Growth curves of individual neo-resistant clones derived on transfection with either NPM/ALK and/or c-myc. G418-resistant clones were isolated 17 days after transfection and further propagated in selection medium. At the time of clonal isolation, cell colonies consisted of 4–20 x 103 cells corresponding to 12–14 population doubling levels (PDL). Clones were passaged in a 1:3 to 1:6 transfer schedule and the cumulative number of PDL was calculated. Cell populations growing for more than 20 PDL are considered immortal and established cell lines. a) Growth of c-myc-transfected clones; b) growth of c-myc/c-H-ras-transfected clones; c) growth of NPM/ALK-transfected clones; d) growth of NPM/ALK/c-myc-transfected clones; e) growth of vector-transfected clones; f) growth of vector/c-H-ras-transfected clones.

2. Prerequisites for NPM/ALK-mediated immortalization
1) Previous reports by others have shown that NPM-mediated hetero-oligomerization of ALK (or leucine zipper-mediated dimerization, respectively) was necessary to transform established 3T3 cells. Leucine zipper proteins, however, frequently form heterodimers with heterologous eukaryotic leucine zipper proteins. Whether homodimerization of ALK was the required functional structure remained unclear. 2) NPM/ALK is ubiquitously expressed in human ALCL cells and in NPM/ALK-expressing REC lines. Correlative data derived from variant chromosomal aberrations sporadically found in ALCL, which also involve truncated ALK, suggested that the oncogenic potential of ALK is exerted by its cytoplasmic localization.

To provide unequivocal data concerning the question of homodimerization and subcellular localization of NPM/ALK, a series of ALK derivatives and fusions were created. Construct NPM/ALK lacks the NPM part and encodes the catalytic domain only. In the TetR/ALK derivative, the NPM portion was substituted by the dimerization domain of bacterial tetracycline repressor (TetR). This dimerization domain warrants actual homodimerization because no known eukaryotic TetR homologue exists. The potential of TetR/ALK to homodimerize was confirmed by coexpressing two TetR/ALK constructs, fused either to a Myc-tag or a V5-tag, and tag-specific immunoprecipitation from native protein lysates in conjunction with Western blot analysis of the alternate tag. In addition, NPM/ALK and TetR/ALK were equipped with a nuclear localization signal (NLS) to specifically express these ALK derivatives in the nucleus, which was confirmed by immunocytochemistry.

Targeting ALK to the nucleus was clearly disadvantageous since biological parameters such as cloning efficiency and immortalization were in the range of vector controls. The ALK monomeric form (without NPM) was inhibitory with respect to cloning efficiency and immortalization. As anticipated, introduction of the TetR homodimerization motif reestablished the oncogenic potential.

3. Cooperation of NPM/ALK and TetR/ALK with c-H-ras
Since NPM/ALK and TetR/ALK immortalized REC, we tested whether either of the constructs might cooperate with c-H-ras in cellular transformation. Approximately one-third of the clones induced by NPM/ALK or TetR/ALK and c-H-ras exhibited a stably transformed morphology, whereas NPM/ALK and NLS-TetR/ALK constructs were far less effective in transformation, most likely due to inefficient c-H-ras cooperation.

4. Cell clones transformed by NPM/ALK and TetR/ALK together with c-H-ras form aggressively growing tumors in syngenic rats
The ultimate proof for malignant transformation of cells is their in vivo tumorigenicity. Thus, four representative lines were injected into 10-day-old syngenic Fisher rats and tumor development was monitored. As expected, no tumors were obtained with a NPM/ALK/neo cell line (clone 249/751) (Fig. 2a ), which confirmed our finding that NPM/ALK alone does not act as a transforming oncogene in normal rat cells. In contrast, the NPM/ALK/ras cell line (clone 249/1024) induced palpable tumor nodules after 7 days in all animals and grew continuously until animals were killed around day 19, when they were still in good condition (Fig. 2b ). Four animals had already developed small tumor nodules 3 days after injection of the transformed TetR/ALK/ras cell lines (clone 239/3711 and clone 239/3741); by day 5, all animals had palpable tumors (Fig. 2c , d ). Tumors grew very rapidly, and by day 13 animals had to be killed because of poor health. ALK overexpression and proper localization were confirmed by immunocytochemistry in all tumor samples.

View larger version (34K): [in this window] [in a new window]   Figure 2. Tumorigenicity of ALK-expressing cell lines. 5 x 105 cells of each cell line were injected subcutaneous (s.c.) into the back of 10-day-old syngenic rats and the growth of tumors was monitored. Each number (A1-A5) represents one animal. a) Parallel lines indicate no tumor outgrowth of this cell line.

CONCLUSIONS AND SIGNIFICANCE

NPM/ALK was previously shown to transform rodent NIH3T3 and FR3T3 cell lines. Undefined genetic alterations have allowed these cells to pass crisis, develop into immortalized cell populations, and might render them susceptible for focus formation as was described for c-myc in FR3T3. Here we demonstrate that NPM/ALK immortalized REC (Fig. 3d ) and only in cooperation with c-H-ras transformed REC (Fig. 3e ), similar to the cooperation of c-myc and c-H-ras (Fig. 3a ). Oligo- (di-)merization of ALK was suggested in previous reports to be necessary for the transforming activity in 3T3 cells, and this was also a prerequisite for the immortalization of REC (Fig. 3f , g ) and tumor formation in syngenic rats (Fig. 3f ). Cytoplasmic localization of ALK dimers is essential for the tumorigenic effect, which agrees with those correlative findings that describe chromosomal aberrations present in ALCL with variant translocations (i.e., in which ALK is translocated to fusion partners other than NPM) and in which ALK occurs strictly in the cytoplasm. It was unexpected to find that the immortalizing effect of ALK was based on its cytoplasmic activity (Fig. 3h ), because immortalizing oncogenes in general encode nuclear proteins. The only known exception is the immortalization of rodent hepatocytes by cytoplasmic c-met receptor tyrosine kinase, which might represent a tissue-specific peculiarity. This report assigns a new and decisive function to NPM/ALK, namely, immortalization. It is discussed that unrestricted growth of leukemic or lymphoid cells could suffice to result in a life-threatening malignancy without the necessity of a series of additional transforming events contributing to tumor cell progression, because blood cells are inherently mobile and ‘invasive’, which is mandatory for their physiological function in the organism.

View larger version (91K): [in this window] [in a new window]   Figure 3. The cooperation of immortalizing and transforming oncogenes in REC. a) Overexpression of c-Myc in the nucleus and of c-H-ras in the cytoplasm, which gives raise to phenotypically transformed clones (cells drawn in pink) and tumor growth in rats. b) c-myc- or d) NPM/ALK overexpression (such as TetR/ALK overexpression; not shown) frequently immortalizes cells (drawn in green), which do not form tumors. c) The majority of cells that overexpress c-H-ras undergo senescence (cells drawn in blue) and have not been shown to form tumors, and thus we have not determined (N.D.) the effect of transiently transformed, yet senescing c-H-ras clones in rats. e) NPM/ALK and c-H-ras or f) TetR/ALK and c-H-ras transform cells and form tumors, whereas g) monomeric NPM/ALK or h) nucleus-targeted NLS-TetR/ALK contribute only marginally to immortalization; in general, clones senesce.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0678fje ; to cite this article, use FASEB J. (April 6, 2001) 10.1096/fj.00-0678fje

² This study is presented in memory of Prof. Dr. Thaddäus Radaszkiewicz.

³ I.S. and D.P. contributed equally to first authorship.

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