Aberrant expression and biological significance of Sox2, an embryonic stem cell transcriptional factor, in ALK-positive anaplastic large cell lymphoma

Abstract

Sox2 (sex-determining region Y-Box) is one of the master transcriptional factors that are important in maintaining the pluripotency of embryonic stem cells (ESCs). In line with this function, Sox2 expression is largely restricted to ESCs and somatic stem cells. We report that Sox2 is expressed in cell lines and tumor samples derived from ALK-positive anaplastic large cell lymphoma (ALK(+)ALCL), for which the normal cellular counterpart is believed to be mature T-cells. The expression of Sox2 in ALK(+)ALCL can be attributed to nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), the oncogenic fusion protein carrying a central pathogenetic role in these tumors. By confocal microscopy, Sox2 protein was detectable in virtually all cells in ALK(+)ALCL cell lines. However, the transcriptional activity of Sox2, as assessed using a Sox2-responsive reporter construct, was detectable only in a small proportion of cells. Importantly, downregulation of Sox2 using short interfering RNA in isolated Sox2(active) cells, but not Sox2(inactive) cells, resulted in a significant decrease in cell growth, invasiveness and tumorigenicity. To conclude, ALK(+)ALCL represents the first example of a hematologic malignancy that aberrantly expresses Sox2, which represents a novel mechanism by which NPM-ALK mediates tumorigenesis. We also found that the transcriptional activity and oncogenic effects of Sox2 can be heterogeneous in cancer cells.

Figure 1

Aberrant expression of Sox2 in ALK⁺ALCL cell lines and patient samples. (a) Reverse-transcription PCR studies demonstrated the expression of Sox2 mRNA (at 294 bp) in three ALK⁺ALCL cell lines. Ntera-2 (a teratocarcinoma cell line) and Hela (a cervical cancer cell line) were used as the positive and negative control, respectively. (b) The Sox2 protein in ALK⁺ALCL cell lines was detectable by western blots. Again, Ntera-2 and Hela cells were used as a positive and negative control, respectively. T-cells isolated from the peripheral blood of healthy donors were negative for Sox2 protein. (c) Nuclear cytoplasmic fractionation experiments showed the nuclear localization of Sox2 in ALK⁺ALCL cells. Ntera-2 and Hela cells were used as a positive and negative control, respectively. (d, e) Confocal immunofluorescence microscopy studies showed that virtually all Karpas 299 and SUP-M2 cells had Sox2 expression, which was localized to the nuclei. (f) Immunohistochemical staining of paraffin-embedded tissue sections revealed that ALK⁺ALCL tumor cells expressed Sox2, which had a predominantly nuclear staining pattern. This was in contrast with the lack of definitive Sox2 staining in benign tonsillar lymphocytes.

Figure 2

Sox2 expression in ALK⁺ALCL can be attributed to NPM-ALK and STAT3. Downregulation of NPM-ALK using specific siRNA decreased the Sox2 protein levels in Karpas 299 (a) and SUP-M2 (b) cells. Similar effects were observed with siRNA downregulation of STAT3 in Karpas 299 (c) and SUP-M2 (d) cells. Results shown are representative of three independent experiments. Of note, cell lysates for this experiment were obtained at 24 and 48 h after the siRNA treatment, and there was no significant cell death observed within this time frame.

Figure 3

Sox2 is transcriptionally active in relatively small subsets of ALK⁺ALCL cell. The percentage of GFP-positive cells in Karpas 299 cells stably transduced with the Sox2 reporter was approximately 10% (a), whereas the percentage of GFP-positive cells in SUP-M2 cells stably transduced with the Sox2 reporter was approximately 30% (b). The results are representative of two different clones for each cell line. Results shown are representative of three independent experiments. (c) To confirm the validity of Sox2 reporter assay, Karpas 299 Sox2^active cells (that is, GFP-positive) were transfected with siRNA targeting Sox2, and we found a substantial decrease in the level of green fluorescence as assessed by flow cytometry. (d) Sox2 expression was confirmed to be dramatically decreased after treatment with siRNA Sox2 by western blotting. Results shown are representative of three independent experiments.

Figure 4

Biochemical comparison of Sox2^active and Sox2^inactive cells. (a, b) By western blots, there was no substantial difference in the total protein levels and the activation/phosphorylation levels of NPM-ALK, STAT3, Akt and p38. However, a slightly higher level of phospho-ERK was found in the Sox2^active cell subset. (c, d) There was no significant difference in cell growth and cell cycle analyses between the Sox2^active and Sox2^inactive subsets derived from Karpas 299 cells. Downregulation of Sox2 expression in ALK⁺ALCL cell lines using siRNA in the Sox2^active cell subset resulted in a significant decrease in cell growth in both Karpas 299 (e) and SUP-M2 (g). Downregulation of Sox2 expression in the Sox2^inactive cell subset using siRNA resulted in no significant change in cell growth in both Karpas 299 (f) and SUP-M2 (h). Results shown are representative of three independent experiments.

Figure 5

Sox2 transcriptional activity correlates with the sensitivity to doxorubicin. Sox2^active subset of cells in both Karpas 299 (a) and SUP-M2 (b) were more resistant to doxorubicin as compared with the Sox2^inactive subset. Results shown are representative of three independent experiments.

Figure 6

Sox2 transcriptional activity correlates with increased invasiveness and contributes to tumorigenicity of ALK⁺ALCL cell lines. Downregulation of Sox2 expression in the Sox2^active subset of Karpas 299 and SUP-M2 cells led to a significant decrease in cell invasiveness (a, c). However, downregulation of Sox2 expression in the Sox2^inactive subset resulted in no significant change (b, d). Furthermore, isolated Karpas 299 Sox2^active cells were significantly more invasive than Sox2^inactive cells (e). Results are expressed in RFU (relative factor unit), which is a measure of cells that have invaded the membrane. The Sox2^active subsets of both Karpas 299 (f) and SUP-M2 (g) showed a significantly higher number of colonies in methylcellulose colony formation assay, as compared with the Sox2^inactive subsets. Data is presented in such a way that the colony numbers of Sox2^inactive subsets were set as 100%. Downregulation of Sox2 expression in the Sox2^active subset of both Karpas 299 (h) and SUP-M2 (j) led to a significant decrease in colony formation. However, downregulation of Sox2 expression in the Sox2^inactive subset resulted in no significant change in both Karpas 299 (i) and SUP-M2 (k). Results shown are representative of three independent experiments.

Figure 7

Sox2 transcriptional activity correlates with tumorogenicity in the SCID xenograft mouse model. Xenografts derived from the Sox2^active subset of SUP-M2 cells were significantly larger than those derived from the Sox2^inactive subset (a, b). A hematoxylin and eosin section of the subcutaneous tumor harvested from an animal xenografted with Sox2^active cells reveals sheets of anaplastic lymphoma cells (c). A high magnification of the splenic tumor from the same animal is illustrated; a megakaryocyte is highlighted by a black arrow (d).