In this study, we have shown that Sox2 expression is a consistent feature of ALK+
ALCL, being detectable in all three cell lines and most of the primary tumors tested. To our knowledge, ALK+
ALCL represents the first example of a hematological malignancy that manifests this feature. In contrast to ALK+
ALCL cells, we did not find Sox2 expression in benign lymphocytes, including those present in peripheral blood and tonsils. Corroborating our findings, a recent study performed by Cimpean et al.40
also found no evidence of Sox2 expression in normal lymphocytes. Nevertheless, it has been reported that Sox2 is expressed in CD34+ hematopoietic stem cells.41, 42
Taken together, these observations suggest that the expression of Sox2 in ALK+
ALCL cells represents an aberrant event. Of note, our immunohistochemical studies demonstrate that Sox2 expression is not limited to ALK+
ALCL in the spectrum of hematological malignancies, as cases of transformed mycosis fungoides and one case of T-cell lymphoma arising in the setting of post-transplantation manifested this phenotype. Further studies are required to confirm whether Sox2 in these T-cell neoplasms without ALK expression is indeed transcriptionally active.
The link between Sox2 and STAT3 has been previously suggested in a study of neural precursor cells.30
Sox2 expression was shown to be upregulated by STAT3, and the promoter region of the Sox2
gene was found to carry multiple STAT3-binding consensus sequences.30
As one of the key characteristics of ALK+
ALCL cells is the constitutive STAT3 activation,23
we hypothesized that the aberrant expression of Sox2 in this cell type is attributed to STAT3. Our data supports this assertion. As the oncogenic tyrosine kinase NPM-ALK is known to be the major activator of STAT3 in ALK+
ALCL, it is not surprising to observe that siRNA downregulation of NPM-ALK also substantially decreased Sox2 expression. In other words, the NPM-ALK/STAT3 signaling axis, which is considered to be the key oncogenic driving force in ALK+
ALCL, is primarily responsible for the aberrant expression of Sox2 in this cancer cell type.
We were rather surprised with the finding that the transcriptional activity of Sox2 is heterogeneous in ALK+ALCL cell lines, as Sox2 protein was expressed in virtually all cells as evidenced by our confocal microscopy experiments. To our knowledge, the observation that the transcriptional activity of Sox2 is heterogeneous in cancer cells has never been previously described. The validity of this novel finding is supported by the following observations. First, ALK+ALCL cell lines stably transduced with the Sox2 reporter construct were cultured in the presence of puromycin selection at all times; thus, it is highly unlikely that the lack of the Sox2 activity in the Sox2inactive subset is due to a loss of the reporter construct. Second, to confirm that the Sox2inactive cells were truly Sox2 inactive, we re-infected these cells with the Sox2 reporter construct, and no increase in GFP expression in these cells was found. Third, using PCR, we were able to detect the presence of the GFP and luciferase genes in the Sox2inactive cells, and thus, the absence of GFP and luciferase expression in these cells is not due to a loss of the reporter. Fourth, the transcriptional activity of Sox2 is associated with different biological characteristics, including cell growth, invasiveness and tumorigenesis. On the basis of our finding that Sox2 transcriptional activity, rather than protein expression, dictates its oncogenic potential, we suggest that future studies investigating Sox2 in cancer should include the transcriptional activity of Sox2 as a major parameter.
An obvious question emerging from our observations is related to how the Sox2 transcriptional activity is regulated. As mentioned above, the differential activity of Sox2 is not due to a difference in Sox2 protein expression, as our confocal microscopy results showed that virtually all cells express this protein. Recently, it was demonstrated that the nuclear localization of Sox2 is regulated by phosphorylation through the AKT signaling.43
Thus, we speculated that the activity of Sox2 is dependent on whether Sox2 is localized to the nuclei where it functions as a transcriptional factor. However, we did not observe any difference in the subcellular localization of Sox2 between the Sox2active
subsets. In another recent study, Van Hoof et al.44
identified that serine phosphorylation of Sox2 can modulate its activity. Again, we did not observe any difference in the serine phosphorylation of Sox2 between the two subsets (Supplementary Figure 10
). Lastly, based on our EMSA results, we observed no obvious difference in the formation of the probe/protein complexes between the two cell subsets. Considering the fact that the transcriptional activity of Sox2 is tightly regulated by a number of co-factors,4, 45
it is likely that one or more of these cofactors are important in regulating the transcriptional activity of Sox2 in ALK+
ALCL cells. In this regard, we investigated the expression of Oct4a, the most studied Sox2 cofactor.5
However, by western blotting, we did not detect any Oct4a expression in ALK+
ALCL cell lines (data not shown). Overall, further studies are needed to delineate how the transcriptional activity of Sox2 is regulated in cancer cells.
In this study, we found that the transcriptional activity of Sox2 correlated with sensitivity to doxorubicin, a conventional chemotherapeutic agent used to treat ALK+
ALCL in the clinic. Interestingly, Mallanna et al.46
have previously reported that Sox2 interacts with DNA repair proteins, such as members of the replication protein A family. Thus, it is possible that the mechanisms that regulate the transcriptional activity of Sox2 may also regulate how Sox2 interacts with DNA repair proteins, thereby modulating the efficiency of DNA repair. Alternatively, some of the downstream targets of Sox2 might be responsible for DNA repair; thus, upregulation of these genes in the Sox2active
subset contributes to enhanced DNA repair and a resistance to doxorubicin.
We have examined the possible mechanisms by which Sox2 exerts its oncogenic effects. We first asked if Sox2 regulates the expression and activation of the NPM-ALK/STAT3 axis. As described, siRNA knockdown of Sox2 in Sox2active
cells did not result in any detectable change to the levels of NPM-ALK, pALK, STAT3 and pSTAT3. The other approach used was to compare the expression and activation status of NPM-ALK and its downstream targets between the Sox2active
cells. A comparison of these two cell subsets showed no substantial difference in the expression/activation status of NPM-ALK, STAT3 and a host of other cellular signaling proteins known to be activated by NPM-ALK. The only exception was p-ERK, which was expressed at a slightly higher level in Sox2active
subset. As described in the results, this increase in p-ERK is not apparently a direct result of Sox2, as siRNA downregulation of Sox2 in the Sox2active
subset did not result in any appreciable change in p-ERK. We also asked if the Sox2 downstream targets in ESCs are differentially expressed between the two cell subsets. As shown in Supplementary Figure 9
, we confirmed the differential expression of the three known Sox2 downstream targets, including notch1, BCL2 and platelet-derived growth factor receptor-α.47, 48
These findings further support our findings of two cell subsets in ALK+
ALCL cells based on their differential Sox2 transcriptional activity. Of interest, both notch1 and platelet-derived growth factor receptor-α have been previously implicated in the pathobiology of ALK+
To conclude, we have demonstrated that ALK+ALCL aberrantly expresses Sox2, one of the master transcriptional factors in ESCs. We have also shown that Sox2 protein expression is dependent on NPM-ALK/STAT3 activation. Interestingly, we have observed that the transcriptional activity of Sox2 is restricted to a relatively small subset of cells, despite the fact that Sox2 protein is expressed in all cells. In view of the fact that the transcriptional activity of Sox2 is critical in mediating tumorigenesis, we believe that further studies investigating how Sox2 activity is regulated will be highly worthwhile.