Background
ALK-positive anaplastic large cell lymphoma (ALK + ALCL) is a specific type of non-Hodgkin lymphoma of null/T cell lineage occurring most frequently in young adults and children [
1,
2]. Approximately 80% of ALK + ALCL patients carry the chromosomal translocation,
t(2;5)(p23;q35), that leads to the generation of the abnormal fusion protein NPM-ALK [
1,
2]. By virtue of its constitutively active tyrosine kinase activity, NPM-ALK drives oncogenesis primarily by binding to and phosphorylating a host of signaling proteins, such as STAT3 and PI3K, thereby deregulating these signaling pathways [
1]. From the clinical perspective, ALK + ALCL tumors are typically aggressive. Complete remission can be induced in most pediatric ALK + ALCL patients with conventional chemotherapy, while chemoresistance and disease relapses occur in a substantial proportion of adult patients [
1]. The biological basis of chemoresistance in ALK + ALCL patients is incompletely understood, but a recent report [
3] describing the existence of cancer stem-like cells (CSCs) raises the possibility that these cells may play a role, similar to how CSCs might contribute to chemoresistance and cancer relapses in other cancer models [
4,
5].
Sox2, one of the four master transcriptional factors involved in re-programming fibroblasts to inducible pluripotent stem cells, is normally expressed in embryonic stem cells [
6]. Recently, aberrant expression of Sox2 has been found in a relatively large number of cancer types, including breast cancer [
7,
8], melanoma [
9], and ALK + ALCL [
10]. Sox2 expression in these cancers has been shown to correlate with cancer stemness properties, such as chemoresistance [
11], tumor initiation [
8,
9], and self-renewal [
9]. Using a Sox2 reporter containing the SRR2 (Sox2 Regulatory Region-2) sequence, we previously identified the existence of two phenotypically distinct cell subpopulations in ALK + ALCL cell lines, with a small subset of cells being Sox2
active (currently denoted as Reporter Responsive, RR) and the majority of the cells being Sox2
inactive (denoted as reporter unresponsive, RU) [
10]. Importantly, the sorted/purified RR cells were found to be significantly more tumorigenic and stem-like compared to their RU counterparts [
10]. Sox2 is directly implicated, since siRNA knockdown of Sox2 resulted in a dramatic abrogation of these features [
10]. As the expression level and subcellular localization of Sox2 were found to be similar between RU and RR cells, we concluded that the RU/RR dichotomy is not a result of a differential Sox2 expression and localization between these two cell subsets [
10]. In view of the link between the RR phenotype and CSC features in ALK + ALCL, we believe that it is of paramount importance to understand the biochemical basis of how the RU/RR dichotomy is regulated.
We hypothesized that Sox2 is more transcriptionally active in RR cells because Sox2 can bind to DNA more efficiently in this cell subset. With this hypothesis, our strategy involved bioinformatics analyses of the SRR2 sequence, in order to identify potential transcriptional factor(s) that regulate the DNA binding of Sox2. With these studies, we identified that a positive feedback loop involving the Wnt/β-catenin/MYC/Sox2 axis defines a highly tumorigenic and chemoresistant cell subset in ALK + ALCL.
Methods
Primary tumors, cell lines, and treatments
All primary tumors were diagnosed at the Cross Cancer Institute (Edmonton, Alberta, Canada), and the diagnostic criteria were based on those described in the WHO classification scheme. The use of these tissues has been approved by our institutional ethics committee. All cell lines were all grown and expanded in RPMI 1640 (Invitrogen, Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS, Invitrogen), 1% penicillin streptomycin (Thermo Fisher Scientific Canada), and 200 ng/mL puromycin dihydrochloride (Sigma-Aldrich, St. Louis, MO) in 5% CO2 atmosphere at 37 °C. Puromycin, G418, 10074-G5, quercetin, doxorubicin, crizotinib, stattic, and iodonitrotetrazolium chloride were all purchased from Sigma-Aldrich. All treatments were performed following the manufacturer’s instructions.
Cell sorting of RU and RR cells
All the RU and RR cells used in this study are sorted and purified RU and RR cells (purity > 95%). Briefly, parental SupM2 and Karpas 299 cells were stably transfected with lentivirus-based SRR2 reporter which contains two readouts including GFP intensity and luciferase activity, as well as puromycin antibiotic marker [
10]. The reporter stably transfected cells were subjected to flow cytometric instrument for cell sorting based on the GFP intensity. The 10% of very GFP-negative cells were sorted as RU cells, and the sorted GFP-positive cells were RR cells. The sorted and purified RU and RR cells were subsequently cultured in cell culture medium with 200 ug/mL puromycin.
Short interfering RNA and transfections
Short interfering RNAs (siRNAs) for MYC, β-catenin, Sox2, and scrambled siRNA were purchased from Dharmacon (Lafayette, CO). Transient transfections of ALK + ALCL cells with siRNAs were performed using the Electro square electroporator BTX ECM 800 (225 V, 8.5 ms, 3 pulses). Briefly, 400 pmol of siRNA were used per 5 million ALK + ALCL cells. The efficiency of target gene inhibition was assessed using western blots.
Luciferase assay
The luciferase assay kit was purchased from Promega (Madison, WI), and luciferase activity was measured following the manufacturer’s protocol.
Transwell assay
The 6-well plates of polyester transwell permeable supports with 0.4-μm pore size were purchased from Corning Inc (Toronto, Ontario, Canada). Briefly, 0.5 million of RU cells and RR cells were seeded in the upper chamber and lower chamber, respectively, and cultured for 72 h. Different ratios of RU/RR cells (RR cells were diluted by RU cells) were also included in this experiment. The same number of RU cells co-cultured with RU cells in the lower chamber was included as control group. Then, the luciferase assay and western blot studies were performed. Note that the upper chamber and lower chamber were seeded with the same number of cells in this experiment.
Western blots
Western blot studies were performed as described previously.2
Antibodies reactive to phosphorylated MYCS62 (E1J4K), MYC (D84C12), Sox2 (D6D9), β-catenin (D10A8), phosphorylated GSK3βS9 (D85E12), LEF1(C18A7), γ-tubulin antibody (#5886), and histone deacetylase 1 (HDAC-1) antibody (#2062) were purchased from Cell Signaling Technology (Danvers, MA); α-tubulin antibody (TU-02), β-catenin (H-102), and β-actin antibody (sc-130300) were purchased from Santa Cruz (Dallas, TX); antibody reactive to active β-catenin (8E7) was purchased from Merck Millipore.
Immunohistochemistry and immunofluorescence studies
Anti-MYC (Y69) antibody (Abcam, Cambridge, MA) was used (1:300 dilution) in the immunohistochemistry assay, following the procedures described previously [
12]. MYC (Y69) antibody (1:300 dilution) and anti-active β-catenin (8E7) antibody (Merck Millipore, Toronto, Ontario, Canada) (1:200 dilution) were used in immunofluorescence double staining. The procedures for the immunofluorescence assay were briefly described as below. Formalin-fixed, paraffin-embedded tissue sections were deparafinized and hydrated. Heat-induced epitope retrieval was performed using citrate buffer (pH = 6) and a pressure cooker using microwave. Tissue sections were then permeabilized for 10 min with 0.2% Triton X-100 in 1× PBS containing 10 mM HEPES and 3% BSA (Sigma-Aldrich), followed by the block with 1× PBS containing 10 mM HEPES and 3% BSA for 1 h. The tissue sections were incubated with primary antibodies reactive to active β-catenin and c-Myc which are diluted in 1× PBS with 10 mM HEPES and 1% BSA overnight in 4 °C. The next day, after three times of washes with 1× PBS (30 min), tissue sections were incubated with secondary antibodies (Alexa Fluor 594 goat anti-rabbit antibody and Alexa Fluor 488 goat anti-mouse antibody, Invitrogen, Burlington, CA), diluted in 1× PBS, 1:300 for 1 h. After washing in 1× PBS, tissues were incubated in 1 μg/mL Hoechst 33342 (Sigma-Aldrich, B2261) for 10 min, followed by washes in 1× PBS and mounted with Mounting Medium (Dako, Mississauga, Ontario, Canada). Cells were visualized with a Zeiss LSM510 confocal microscope (Carl Zeiss, Heidelberg, Germany) at the Core Cell Imaging Facility, Cross Cancer Institute, University of Alberta, Edmonton, Canada.
SRR2 probe binding assay
Cells were harvested and washed with cold PBS twice, following by cytoplasmic and nuclear fractionation using the Pierce NE-PER kit (Fisher Scientific Canada). Three hundred micrograms of nuclear proteins was incubated with or without 3 pmol of biotin-labeled SRR2 probe (constructed by IDT, Edmonton, Alberta, Canada) for 0.5 h by rotating at room temperature. Streptavidin agarose beads (75 μL, Fisher Scientific) were added to each sample, following by overnight rotation at 4 °C. The next day, the samples were washed with cold PBS three times for 30 min in total, and protein was eluted at 100 °C in 4X protein loading buffer for 5 min, followed by western blot study.
The sequence of the SRR2 probe: 5′-AAGAATTTCCCGGGCTCGGGCAGCCATTGTGATGCATATAGGATTATTCACGTGGTAATG-3′
The underlined sequence is the Sox2 consensus sequence.
SCID mouse xenograft studies
Twelve CB-17 strain SCID male mice, purchased from Taconic (Hudson, NY), were housed in a virus- and antigen-free facility supported by the Health Sciences Laboratory Animal Services at the University of Alberta and were cared for in accordance with the Canadian Council on Animal Care guidelines. All experimental protocols involving mice were reviewed and approved by the University of Alberta Health Sciences Animal Welfare Committee. Briefly, 2 million cells of SupM2-RU-EV, SupM2-RU-MYC, and SupM2-RR-EV growing exponentially were injected into both flanks of 4-week-old mice, four mice each group. The tumor sizes were measured twice every week. These animals were sacrificed when a tumor reached 10 mm in the greatest dimension.
Statistical analysis
Data is expressed as mean ± standard deviation. The statistical analysis was performed using GraphPad Prism 5 (La Jolla, CA), and the significance of two independent groups of samples was determined using Student’s
t test. Statistical significance is denoted by * (
P < 0.05) and ** (
P < 0.01). For additional methods, see Additional file
1.
Discussion
One of the key findings of this study is that MYC appears to be the key regulator of the RU/RR dichotomy in ALK + ALCL. Our study showed that MYC is crucial for the SRR2 activity and the RR phenotype, since knockdown of MYC by siRNA or pharmacological agent in RR cells abolishes the SRR2 activity and the RR phenotype. Importantly, we found evidence that the regulatory function of MYC is related to its ability to influence the DNA binding and transcriptional activity of Sox2. This model explains why RU and RR cells have dramatically different SRR2 activity despite their approximately equal Sox2 protein expression level and nuclear localization. As illustrated in Fig.
5c, a relatively high level of MYC, perhaps exceeding a specific threshold, permits the binding of Sox2 to SRR2 and its execution as a transcriptional factor. To our knowledge, this is the first report describing this novel relationship between MYC and Sox2 in cancer cells. Exactly how a high level of MYC promotes the DNA binding of Sox2 is unknown. A recently published observation [
21] that the target genes of MYC substantially overlap with those of Sox2 suggests that MYC or the MYC protein complex may physically direct Sox2 to the gene promoters, and facilitate its DNA binding. This concept is supported by a previously published data that MYC and Sox2 were found co-localized in a protein complex [
22]. Furthermore, MYC has been recently reported to regulate gene expression as a general transcriptional amplifier [
23,
24].
MYC, one of the four inducible pluripotent stem cell factors [
25], is known to contribute to cancer stemness in cancer cells, including the tumor-initiating ability [
26,
27], chemoresistance [
28,
29], and self-renewal [
30]. Moreover, the expression level of MYC was found to be relatively high in CSCs derived from several cancer types when compared to bulk cell populations [
30‐
34]. A recent study has highlighted that the CSCs from glioma are more sensitive than the bulk tumor cells to cell death induced by MYC inhibition [
30]. This observation correlates well with our finding that RR cells derived from ALK + ALCL are more sensitive to cell growth inhibition induced by MYC inhibition, as compared to RU cells. How exactly MYC mediates these biological effects is not completely understood, but it is believed that MYC can regulate as many as ~15% of human genes that are involved in critical cellular processes including chromatin remodeling, cell cycle control, metabolism, and self-renewal [
27,
35]. Importantly, MYC is found to bind to and regulate
SOX2 gene expression in CSCs derived from triple negative breast cancer, suggesting MYC can regulate cancer stemness by modulating the expression of other critical embryonic stem cell markers such as Sox2 [
36]. With this context, we believe that findings of this study have advanced by our understanding of how MYC may promote stem-like features, namely by enhancing Sox2/DNA binding and Sox2 transcriptional activity.
The role of MYC in ALK + ALCL has not been extensively studied, and we are aware of only two publications that directly studied MYC in these tumors. In a recent study, shRNA knockdown of MYC was found to reduce the growth of ALK + ALCL cells in vitro, although the underlying mechanism was not delineated [
37]. Consistent with this observation, we also found that pharmacologic inhibition of MYC can significantly inhibit the growth in ALK + ALCL cells. Importantly, we found that RR cells were found to be more sensitive to MYC inhibition than RU cells, consistent with our model that MYC carries more biological importance in RR cells. The second study published described that MYC in ALK + ALCL can be upregulated by NPM-ALK [
38], but the biological significance of this observation was not assessed. Correlating with this finding, we also found that NPM-ALK upregulates MYC. However, while the NPM-ALK/STAT3 axis contributes to the expression of MYC in both RU and RR cells, this pathway is not responsible for the differential MYC expression between these two cell subsets.
As the NPM-ALK/STAT3 signaling pathway is not the key contributing factor to the differential expression of MYC, we turned to the Wnt/β-catenin pathway, which has been well documented to upregulate MYC in a variety of human cancers [
15‐
17]. Constitutive activation of the Wnt/β-catenin pathway can be found in CSCs derived from various cancer types [
39‐
42]. Inhibition of the Wnt/β-catenin pathway has been shown to decrease stemness and tumorigenic potential in cancer cells [
39‐
42], and there is evidence that MYC is a mediator of the stemness properties conferred by this pathway [
43,
44]. A recent study suggested that MYC is the ultimate downstream of β-catenin pathway-mediated enhanced amplification and tumorigenesis of basal stem cells [
44]. Our model is in line with these observations, although our model highlights the importance of intra-tumoral heterogeneity and suggests that a high activation level of the Wnt/β-catenin pathway is a characteristic of RR cells. While this concept has been brought up in a previous publication [
41], our data has provided the mechanistic explanation as to how the Wnt/β-catenin pathway may promote stemness for the first time. Specifically, a high level of Wnt/β-catenin activity promotes a relatively high level of MYC expression, which permits Sox2 to exert its transcriptional activity. Furthermore, based on our observations that Sox2 upregulates a number of Wnt/β-catenin pathway ligands as well as β-catenin, we have demonstrated, for the first time, a positive feedback loop involving Wnt/β-catenin, MYC, and Sox2, and our hypothetical model has been illustrated in Fig.
5c. Our data supports a model in which this positive feedback loop is the defining feature of RR cells in ALK + ALCL. Here, we need to stress that our observation that blocking either NPM-ALK/STAT3 or Wnt/β-catenin nearly diminished the expression of MYC in RR cells seems against our hypothetical model. While we are aware that the published literature [
45,
46] demonstrating the reciprocal regulation between NPM-ALK/STAT3 and Wnt/β-catenin in ALK + ALCL can help explain our observation. In other words, suppression of one would attenuate the activity of the other one.
Results from our immunofluorescence staining/confocal microscopy have provided further evidence to support the existence of the positive feedback loop involving Wnt/β-catenin and MYC in a small cell subset of ALK + ALCL. Thus, MYC significantly co-localizes with active β-catenin in a very small number of tumor cells. Regarding our immunohistochemical studies, we would like to point out that, while we found only ~30% of tumor cells being labeled with MYC, two previous publications showed that MYC immunohistochemical reactivity is detectable in the majority of tumor cells in ALK + ALCL [
37,
38]. This discrepancy may be due to the use of different MYC antibodies and/or immunostaining protocols. In our experience, a substantially higher number of MYC-positive cells can be obtained if higher concentration of anti-MYC antibody is used. The concentration of anti-MYC antibody we chose was based on the observation that this antibody concentration is optimal in revealing the intra-tumoral heterogeneity of MYC expression.
Acknowledgements
The authors would like to thank the technical assistance of Gareth Palidwor, University of Ottawa, in the in silico JASPAR motif match analyses. The authors also would like to thank Yuen Morrissey, University of Alberta, for reading the manuscript.