Oxidative stress enhances tumorigenicity and stem-like features via the activation of the Wnt/?-catenin/MYC/Sox2 axis in ALK-positive anaplastic large-cell lymphoma.
ABSTRACT: The phenomenon that malignant cells can acquire stemness under specific stimuli, encompassed under the concept of cancer cell plasticity, has been well-described in epithelial malignancies. To our knowledge, cancer cell plasticity has not yet been described in hematopoietic cancers. To illustrate and study cancer cell plasticity in hematopoietic cancers, we employed an in-vitro experimental model of ALK-positive anaplastic large-cell lymphoma (ALK+ALCL) that is based on the phenotypic and functional dichotomy of these cells, with cells responsive to a Sox2 reporter (i.e. RR cells) being significantly more stem-like than those unresponsive to the reporter (i.e. RU cells).H2O2 was employed to trigger oxidative stress. GFP expression and luciferase activity, readouts of the Sox2 reporter activity, were quantified by using flow cytometry and luciferase activity assay, respectively. Doxorubicin-resistance and clonogenicity were assessed by using the MTS, methylcellulose colony formation and limiting dilution assays. Western blotting and quantitative PCR were used to assess the expression of various members of the Wnt/?-catenin pathway. Pull-down studies using a Sox2 binding consensus sequence were used to assess Sox2-DNA binding. Quercetin and 10074-G5 were used to inhibit ?-catenin and MYC, respectively. siRNA was used to downregulate Sox2.Under H2O2-induced oxidative stress, a substantial fraction of RU cells was found to convert to RR cells, as evidenced by their acquisition of GFP expression and luciferase activity. Compared to the native RU cells, converted RR cells had significantly higher levels of doxorubicin-resistance, clonogenicity and sphere formation. Converted RR cells were characterized by an upregulation of the Wnt/?-catenin/MYC/Sox2 signaling axis, previously found to be the key regulator of the RU/RR dichotomy in ALK+ALCL. Furthermore, Sox2 was found to bind to DNA efficiently in converted RR cells but not RU cells, and this finding correlated with significant elevations of several Sox2 downstream targets such as WNT2B and BCL9. Lastly, inhibition of ?-catenin, MYC or Sox2 in RU cells significantly abrogated the H2O2-induced RU/RR conversion.We have demonstrated that cancer cell plasticity exists in ALK+ALCL, a type of hematopoietic cancer. In this cancer type, the Wnt/?-catenin/MYC/Sox2 axis is an important regulator of cancer cell plasticity.
Project description:ALK has been identified as a novel therapeutic target in neuroblastoma (NB), but resistance to ALK inhibitors (such as crizotinib) is well recognized. We recently published that the crizotinib sensitivity in NB cells strongly correlates with the crizotinib-ALK binding, and ?-catenin effectively hinders this interaction and confers crizotinib resistance. Here, we asked if these observations hold true for the stem-like cells in NB cells, which were purified based on their responsiveness to a Sox2 reporter. Compared to bulk, reporter unresponsive (RU) cells, reporter responsive (RR) cells had significantly higher neurosphere formation ability, expression of CD133/nestin and chemo-resistance. Using the cellular thermal shift assay, we found that RR cells exhibited significantly weaker crizotinib-ALK binding and higher crizotinib resistance than RU cells. The suboptimal crizotinib-ALK binding in RR cells can be attributed to their high ?-catenin expression, since siRNA knockdown of ?-catenin restored the crizotinib-ALK binding and lowered the crizotinib resistance to the level of RU cells. Enforced expression of ?-catenin in RU cells resulted in the opposite effects. To conclude, high expression of ?-catenin in the stem-like NB cells contributes to their crizotinib resistance. Combining ?-catenin inhibitors and ALK inhibitors may be useful in treating NB patients.
Project description: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.
Project description:We have previously identified a novel intra-tumoral dichotomy in breast cancer based on the differential responsiveness to a Sox2 reporter (SRR2), with cells responsive to SRR2 (RR) being more stem-like than unresponsive cells (RU). Here, we report that RR cells derived from MCF7 and ZR751 displayed a higher tolerance to oxidative stress than their RU counterparts, supporting the concept that the RR phenotype correlates with cancer stemness. Sox2 is directly implicated in this differential H2O2 tolerance, since siRNA knockdown of Sox2 in RR cells leveled this difference. Interestingly, H2O2 converted a proportion of RU cells into RR cells, as evidenced by their expression of luciferase and GFP, markers of SRR2 activity. Compared to RU cells, converted RR cells showed a significant increase in mammosphere formation and tolerance to H2O2. Converted RR cells also adopted the biochemical features of RR cells, as evidenced by their substantial increase in Sox2-SRR2 binding and the expression of 3 signature genes of RR cells (CD133, GPR49 and MUC15). Lastly, the H2O2-induced RU/RR conversion was detectable in a SCID mouse xenograft model and primary tumor cells. To conclude, the H2O2-induced RU/RR conversion has provided a novel model to study the acquisition of cancer stemness and plasticity.
Project description:We have previously identified a novel intra-tumoral dichotomy in triple-negative breast cancer (TNBC) based on the differential responsiveness to a reporter containing the Sox2 regulatory region-2 (SRR2), with reporter responsive (RR) cells being more stem-like than reporter unresponsive (RU) cells. Using bioinformatics, we profiled the protein-DNA binding motifs of SRR2 and identified Myc as one of the potential transcription factors driving SRR2 activity. In support of its role, Myc was found to be highly expressed in RR cells as compared to RU cells. Enforced expression of MYC in RU cells resulted in a significant increase in SRR2 activity, Myc-DNA binding, proportion of cellsexpressing CD44+/CD24-, chemoresistance and mammosphere formation. Knockdown of Myc using siRNA in RR cells led to the opposite effects. We also found evidence that the relatively high ERK activation in RR cells contributes to their high expression of Myc and stem-like features. Using confocal microscopy and patient samples, we found a co-localization between Myc and CD44 in the same cell population. Lastly, a high proportion of Myc-positive cells in tumors significantly correlated with a short patient survival. In conclusion, inhibition of the MAPK/ERK/Myc axis may be an effective approach in eliminating stem-like cells in TNBC.
Project description:We found that MCF7 and ZR751 Sox2-expressing breast cancer cell lines comprise of cells with heterogeneous Sox2 transcription activity reporter response. A small subset of Sox2 reporter responsive cells are more tumourigenic than the bulk Sox2 reporter unresponsive cells. We questioned whether Sox2 exhibit differential gene promoter occupancies in the two cell subsets to govern differential gene expression patterns. Overall design: Sox2 ChIP in reporter unresponsive (RU) and reporter responsive (RR) cells (duplicate samples) were compared. IgG ChIP in RU and RR cells served as the negative controls.
Project description:Although anaplastic large-cell lymphomas (ALCL) carrying anaplastic lymphoma kinase (ALK) have a relatively good prognosis, aggressive forms exist. We have identified a novel translocation, causing the fusion of the TRAF1 and ALK genes, in one patient who presented with a leukemic ALK+ ALCL (ALCL-11). To uncover the mechanisms leading to high-grade ALCL, we developed a human patient-derived tumorgraft (hPDT) line. Molecular characterization of primary and PDT cells demonstrated the activation of ALK and nuclear factor kB (NFkB) pathways. Genomic studies of ALCL-11 showed the TP53 loss and the in vivo subclonal expansion of lymphoma cells, lacking PRDM1/Blimp1 and carrying c-MYC gene amplification. The treatment with proteasome inhibitors of TRAF1-ALK cells led to the downregulation of p50/p52 and lymphoma growth inhibition. Moreover, a NFkB gene set classifier stratified ALCL in distinct subsets with different clinical outcome. Although a selective ALK inhibitor (CEP28122) resulted in a significant clinical response of hPDT mice, nevertheless the disease could not be eradicated. These data indicate that the activation of NFkB signaling contributes to the neoplastic phenotype of TRAF1-ALK ALCL. ALCL hPDTs are invaluable tools to validate the role of druggable molecules, predict therapeutic responses and implement patient specific therapies.
Project description:We found that MCF7 and ZR751 Sox2-expressing breast cancer cell lines comprise of cells with heterogeneous Sox2 transcription activity reporter response. A small subset of Sox2 reporter responsive cells are more tumourigenic than the bulk Sox2 reporter unresponsive cells. We questioned whether Sox2 exhibit differential gene promoter occupancies in the two cell subsets to govern differential gene expression patterns. Sox2 ChIP in reporter unresponsive (RU) and reporter responsive (RR) cells (duplicate samples) were compared. IgG ChIP in RU and RR cells served as the negative controls.
Project description:Anaplastic large cell lymphomas (ALCL) represent a peripheral T-cell lymphoma subgroup, stratified based on the presence or absence of anaplastic lymphoma kinase (ALK) chimeras. Although ALK-positive ALCLs have a more favorable outcome than ALK-negative ALCL, refractory and/or relapsed forms are common and novel treatments are needed. Here we investigated the therapeutic potential of a novel bromodomain inhibitor, OTX015/MK-8628 in ALK-positive ALCLs.The effects of OTX015 on a panel of ALK+ ALCL cell lines was evaluated in terms of proliferation, cell cycle and downstream signaling, including gene expression profiling analyses. Synergy was tested with combination targeted therapies.Bromodomain inhibition with OTX015 led primarily to ALCL cell cycle arrest in a dose-dependent manner, along with downregulation of MYC and its downstream regulated genes. MYC overexpression did not compensate this OTX015-mediated phenotype. Transcriptomic analysis of OTX015-treated ALCL cells identified a gene signature common to various hematologic malignancies treated with bromodomain inhibitors, notably large cell lymphoma. OTX015-modulated genes included transcription factors (E2F2, NFKBIZ, FOS, JUNB, ID1, HOXA5 and HOXC6), members of multiple signaling pathways (ITK, PRKCH, and MKNK2), and histones (clusters 1-3). Combination of OTX015 with the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib led to cell cycle arrest then cell death, and combination with suboptimal doses of the ALK inhibitor CEP28122 caused cell cycle arrest. When OTX015 was associated with GANT61, a selective GLI1/2 inhibitor, C1156Y-resistant ALK ALCL growth was impaired.These findings support OTX015 clinical trials in refractory ALCL in combination with inhibitors of interleukin-2-inducible kinase or SHH/GLI1.
Project description:We have previously identified a novel phenotypic dichotomy in breast cancer (BC) based on the response to a SRR2 (Sox2 regulatory region 2) reporter, with reporter responsive (RR) cells being more tumorigenic/stem-like than reporter unresponsive (RU) cells. Since the expression level of Sox2 is comparable between the two cell subsets, we hypothesized that post-translational modifications of Sox2 contribute to their differential reporter response and phenotypic differences. By liquid chromatography-mass spectrometry, we found Sox2 to be phosphorylated in RR but not RU cells. Threonine 116 is an important phosphorylation site, since transfection of the T116A mutant into RR cells significantly decreased the SRR2 reporter luciferase activity and the RR-associated phenotype. Oxidative stress-induced conversion of RU into RR cells was accompanied by Sox2 phosphorylation at T116 and increased Sox2-DNA binding. In a cohort of BC, we found significant correlations between the proportion of tumor cells immuno-reactive with anti-phosphorylated Sox2T116 and a high tumor grade (p = 0.006), vascular invasion (p = 0.001) and estrogen receptor expression (p = 0.032). In conclusion, our data suggests that phosphorylation of Sox2T116 contributes to the tumorigenic/stem-like features in RR cells. Detection of phospho-Sox2T116 may be useful in identifying a small subset of tumor cells carrying stem-like/tumorigenic features in BC.
Project description:Sox2, a transcription factor and an embryonic stem cell marker, has been implicated in the pathogenesis of breast cancer (BC). YB-1 is another transcription factor that has been shown to promote stemness in BC cells.Western blotting, quantitative PCR, and siRNAs were used to query the regulatory relationships between YB-1, Sox2, and their downstream targets. Chromatin immunoprecipitation was used to detect YB-1 interactions at the Sox2 promoter. Mammosphere and soft agar assays were used to assess the phenotypic consequences of YB-1 knockdown.Here, we report that YB-1 regulates Sox2. YB-1 was found to bind to the SOX2 promoter and down-regulate its expression in MCF7 and ZR751. The regulatory interaction between YB-1 and Sox2 was drastically different between the two phenotypically distinct cell subsets, purified based on their differential response to a Sox2 reporter. They are referred to as the reporter unresponsive (RU) cells and the reporter responsive (RR) cells. Upon siRNA knockdown of YB-1, RU cells showed an increase in Sox2 expression but no change in Sox2 reporter activity; in contrast, RR cells exhibited increased expression and reporter activity of Sox2. Correlating with these findings, YB-1 knockdown induced a differential response in the expression of genes known to be regulated by both Sox2 and YB-1 (e.g. CCND1 and ITGA6). For instance, in response to YB-1 knockdown, CCND1 and ITGA6 expression were decreased or unchanged in RU cells but paradoxically increased in RR cells. Compared to RU cells, RR cells were significantly more resistant to the suppression of mammosphere formation due to YB-1 knockdown. Importantly, mammospheres derived from parental MCF7 cells treated with YB-1 siRNA knockdown exhibited higher expression levels of SOX2 and its downstream targets.To conclude, in a subset of BC cells, namely RR cells, YB-1 regulates Sox2 to coordinately maintain stemness and tumorigenic properties.