Gene Methylation and Silencing of WIF1 Is a Frequent Genetic Abnormality in Mantle Cell Lymphoma.

We have previously shown that the Wnt canonical pathway (WCP) is constitutively active in most cases of mantle cell lymphoma (MCL). Here, we aimed to elucidate the mechanisms underlying this biochemical deregulation. We hypothesized that gene methylation/silencing of WIF1 (Wnt inhibitory factor-1), a physiologic inhibitor of WCP, contributes to the deregulation of WCP and promotes cell growth in MCL. In support of this hypothesis, we found that the expression of WIF1 was detectable in none of the 4 MCL cell lines, and in only 2 of 5 tumors (40%) examined. Using methylation-specific PCR, we found evidence of gene methylation of WIF1 in 4 of 5 cell lines (80%) and in 24 of 29 (82%) tumors. The addition of the demethylation agent 5-aza-2'-deoxycytidine to Mino and JeKo-1, two WIF1-negative cell lines, restored the expression of WIF1 mRNA in these cells. Gene transfection of WIF1 into JeKo-1 and Mino cells significantly reduced cell growth, and this finding correlated with substantial downregulations of various proteins in WCP, such as ß-catenin and pGSK-3ß. In conclusion, our results support the concept that gene methylation/silencing of WIF1 is a frequent event in MCL, and this abnormality contributes to the aberrant activation of WCP. These results have provided further evidence that aberrant Wnt signaling is pathogenetically important in MCL and it may represent a potential therapeutic target.

Oxidative stress enhances tumorigenicity and stem-like features via the activation of the Wnt/ß-catenin/MYC/Sox2 axis in ALK-positive anaplastic large-cell lymphoma.

BACKGROUND: 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). METHODS: 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/ß-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 ß-catenin and MYC, respectively. siRNA was used to downregulate Sox2. RESULTS: 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/ß-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 ß-catenin, MYC or Sox2 in RU cells significantly abrogated the H2O2-induced RU/RR conversion. CONCLUSIONS: We have demonstrated that cancer cell plasticity exists in ALK+ALCL, a type of hematopoietic cancer. In this cancer type, the Wnt/ß-catenin/MYC/Sox2 axis is an important regulator of cancer cell plasticity.

STAT1 is phosphorylated and downregulated by the oncogenic tyrosine kinase NPM-ALK in ALK-positive anaplastic large-cell lymphoma.

The tumorigenicity of most cases of ALK-positive anaplastic large-cell lymphoma (ALK+ ALCL) is driven by the oncogenic fusion protein NPM-ALK in a STAT3-dependent manner. Because it has been shown that STAT3 can be inhibited by STAT1 in some experimental models, we hypothesized that the STAT1 signaling pathway is defective in ALK+ ALCL, thereby leaving the STAT3 signaling unchecked. Compared with normal T cells, ALK+ ALCL tumors consistently expressed a low level of STAT1. Inhibition of the ubiquitin-proteasome pathway appreciably increased STAT1 expression in ALK+ ALCL cells. Furthermore, we found evidence that NPM-ALK binds to and phosphorylates STAT1, thereby promoting its proteasomal degradation in a STAT3-dependent manner. If restored, STAT1 is functionally intact in ALK+ ALCL cells, because it effectively upregulated interferon-γ, induced apoptosis/cell-cycle arrest, potentiated the inhibitory effects of doxorubicin, and suppressed tumor growth in vivo. STAT1 interfered with the STAT3 signaling by decreasing STAT3 transcriptional activity/DNA binding and its homodimerization. The importance of the STAT1/STAT3 functional interaction was further highlighted by the observation that short interfering RNA knockdown of STAT1 significantly decreased apoptosis induced by STAT3 inhibition. Thus, STAT1 is a tumor suppressor in ALK+ ALCL. Phosphorylation and downregulation of STAT1 by NPM-ALK represent other mechanisms by which this oncogenic tyrosine kinase promotes tumorigenesis.

The PI3K/AKT/c-MYC Axis Promotes the Acquisition of Cancer Stem-Like Features in Esophageal Squamous Cell Carcinoma.

The importance of intratumoral heterogeneity has been highlighted by the identification and characterization of cancer stem cells (CSCs). Based on the differential responsiveness to a Sox2 reporter, SRR2, we had found a novel dichotomy in esophageal squamous cell carcinoma (ESCC) cells, with reporter-responsive (RR) cells showing more CSC-like features than reporter-unresponsive (RU) cells. Specifically, RR cells exhibited significantly higher tumorsphere formation capacity, proportions of CD44(High) cells, chemoresistance to cisplatin, and tumorigenic potential in vivo. H2 O2 , a potent inducer of oxidative stress and reactive oxygen species, was found to induce a conversion from RU to RR cells; importantly, converted RR cells acquired CSC-like features. The PI3K/AKT/c-MYC signalling axis is important in this context, since pharmacologic blockade of PI3K-AKT or siRNA knockdown of c-MYC effectively inhibited the RR phenotype and its associated CSC-like features, as well as the H2 O2 -induced RU/RR conversion. In a cohort of 188 ESCC patient samples, we found a significant correlation between strong c-MYC expression and a short overall survival (p = .009). In conclusion, we have described a novel intratumoral heterogeneity in ESCC. The identification of the PI3K/AKT/c-MYC axis as a driver of CSC-like features carries therapeutic implications. Stem Cells 2016;34:2040-2051.

miR-200b induces cell cycle arrest and represses cell growth in esophageal squamous cell carcinoma.

miR-200b is a pleiotropically acting microRNA in cancer progression, representing an attractive therapeutic target. We previously identified miR-200b as an invasiveness repressor in esophageal squamous cell carcinoma (ESCC), whereas further understanding is warranted to establish it as a therapeutic target. Here, we show that miR-200b mitigates ESCC cell growth by inducing G2-phase cell cycle arrest and apoptosis. The expression/activation of multiple key cell cycle regulators such as CDK1, CDK2, CDK4 and Cyclin B, and the Wnt/ß-Catenin signaling are modulated by miR-200b. We identified CDK2 and PAF (PCNA-associated factor), two important tumor-promoting factors, as direct miR-200b targets in ESCC. Correlating with the frequent loss of miR-200b in ESCC, both CDK2 and PAF levels are significantly increased in ESCC tumors compared to case-matched normal tissues (n = 119, both P < 0.0001), and correlate with markedly reduced survival (P = 0.007 and P = 0.041, respectively). Furthermore, CDK2 and PAF are also associated with poor prognosis in certain subtypes of breast cancer (n = 1802) and gastric cancer (n = 233). Although CDK2 could not significantly mediate the biological function of miR-200b, PAF siRNA knockdown phenocopied while restored expression of PAF abrogated the biological effects of miR-200b on ESCC cells. Moreover, PAF was revealed to mediate the inhibitory effects of miR-200b on Wnt/ß-Catenin signaling. Collectively, the pleiotropic effects of miR-200b in ESCC highlight its potential for therapeutic intervention in this aggressive disease.

Profiling gene promoter occupancy of Sox2 in two phenotypically distinct breast cancer cell subsets using chromatin immunoprecipitation and genome-wide promoter microarrays.

INTRODUCTION: Aberrant expression of the embryonic stem cell marker Sox2 has been reported in breast cancer (BC). We previously identified two phenotypically distinct BC cell subsets separated based on their differential response to a Sox2 transcription activity reporter, namely the reporter-unresponsive (RU) and the more tumorigenic reporter-responsive (RR) cells. We hypothesized that Sox2, as a transcription factor, contributes to their phenotypic differences by mediating differential gene expression in these two cell subsets. METHODS: We used chromatin immunoprecipitation and a human genome-wide promoter microarray (ChIP-chip) to determine the promoter occupancies of Sox2 in the MCF7 RU and RR breast cancer cell populations. We validated our findings with conventional chromatin immunoprecipitation, quantitative reverse transcription polymerase chain reaction (qPCR), and western blotting using cell lines, and also performed qPCR using patient RU and RR samples. RESULTS: We found a largely mutually exclusive profile of gene promoters bound by Sox2 between RU and RR cells derived from MCF7 (1830 and 456 genes, respectively, with only 62 overlapping genes). Sox2 was bound to stem cell- and cancer-associated genes in RR cells. Using quantitative RT-PCR, we confirmed that 15 such genes, including PROM1 (CD133), BMI1, GPR49 (LGR5), and MUC15, were expressed significantly higher in RR cells. Using siRNA knockdown or enforced expression of Sox2, we found that Sox2 directly contributes to the higher expression of these genes in RR cells. Mucin-15, a novel Sox2 downstream target in BC, contributes to the mammosphere formation of BC cells. Parallel findings were observed in the RU and RR cells derived from patient samples. CONCLUSIONS: In conclusion, our data supports the model that the Sox2 induces differential gene expression in the two distinct cell subsets in BC, and contributes to their phenotypic differences.

Cancer Research Challenges and Potential Solutions in Saudi Arabia: A Qualitative Discussion Group Study.

PURPOSE: Cancer incidence in Saudi Arabia has recently shown an upward trend. Research efforts within the different cancer continuum are pivotal to strengthening control measures. Since cancer research is evolving in the country, it is crucial to understand the current challenges and implement defined interventions to overcome them. The present qualitative study aimed to assess cancer research barriers among researchers and identify potential solutions from their perspectives. METHODS: We conducted a focus group discussion among 17 Saudi-based cancer researchers from diverse research backgrounds, provinces, and institutions. We used descriptive-interpretive thematic analysis following an open-ended approach to investigate the challenges in conducting cancer research. We also captured the solutions suggested based on the researchers' experiences. RESULTS: Six major themes emerged from the analysis: requirements of the data landscape, organizational support, national research roadmap, sustainable funding, clearer policies and regulations, and capacity building. To address challenges in these areas, researchers stressed the need for improved interinstitutional collaborations, immediate availability of research materials, and unlimited and easy access to research data. CONCLUSION: Improving health research is one of the primary goals of Saudi Vision 2030. It is, therefore, essential to overcome the current challenges in cancer research, enabling research findings to inform policies related to cancer control and care provision.

Effect of SARS-CoV-2 Entry Factors on Myeloid Cancers.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel, highly pathogenic coronavirus that has spread rapidly worldwide and caused an international public health emergency. Patients with hematological cancers are regarded as a high-risk group for coronavirus disease 2019 (COVID-19). However, few reports have investigated factors that might account for the differential severity of COVID-19 disease in these patients. METHODS: Gene expression of SARS-CoV-2 entry-promoting factors and entry-restricting factors and the associated effects on myeloid malignancies were evaluated. Gene expression levels of 11 SARS-CoV-2 entry-promoting factors and 4 SARS-CoV-2 entry-restricting factors were analyzed in patients with myelodysplastic syndromes (MDS), chronic myeloid leukemia (CML), and acute myeloid leukemia and its subtypes. RESULTS: Expression levels of promoting and restricting factors were most affected in MDS. Specifically, 4 of the 11 analyzed SARS-CoV-2 entry-promoting factors were significantly increased (TMPRSS4, CD209, CLEC4G, and CTSB), and 2 of the 4 analyzed SARS-CoV-2 entry-restricting factors were significantly decreased (IFITM1 and IFITM2) in MDS. Patients with CML also exhibited a pattern of significant changes in SARS-CoV-2 entry-promoting and entry-restricting factors. Five of the 11 analyzed SARS-CoV-2 entry-promoting factors were significantly increased (ACE2, TMPRSS2, TMPRSS4, ANPEP, CD209), and 1 of the 4 analyzed SARS-CoV-2 entry-restricting factors was significantly decreased (LY6E) in CML. CONCLUSIONS: The present and past results highlight the importance of investigating SARS-CoV-2 entry-promoting factors and entry-restricting factors, because of their crucial role in determining the differential severity of COVID-19 disease.

Rare loss-of-function variants in type I IFN immunity genes are not associated with severe COVID-19.

A recent report found that rare predicted loss-of-function (pLOF) variants across 13 candidate genes in TLR3- and IRF7-dependent type I IFN pathways explain up to 3.5% of severe COVID-19 cases. We performed whole-exome or whole-genome sequencing of 1,864 COVID-19 cases (713 with severe and 1,151 with mild disease) and 15,033 ancestry-matched population controls across 4 independent COVID-19 biobanks. We tested whether rare pLOF variants in these 13 genes were associated with severe COVID-19. We identified only 1 rare pLOF mutation across these genes among 713 cases with severe COVID-19 and observed no enrichment of pLOFs in severe cases compared to population controls or mild COVID-19 cases. We found no evidence of association of rare LOF variants in the 13 candidate genes with severe COVID-19 outcomes.

Clinico-Pathological Study of K-ras Mutations in Colorectal Tumors: A Single-Center Retrospective Study of 51 Patients in Madinah, Saudi Arabia.

Background Colorectal cancer (CRC) is one of the leading types of cancer worldwide and in Saudi Arabia. At the molecular level, CRC is very complicated and requires establishing comprehensive patient stratification models through identification of patients who will benefit or will not benefit from targeted therapy. We retrospectively investigated and analyzed the frequency of Kirsten-ras (K-ras) mutation and its correlation with patients' characteristics as weel as its association with clinicopathological features (i.e age, gender, clinical stage, anatomical site, histological subtype, degree of histological differentiation and metastatic site) in patients with CRC. Methods Medical records and paraffin-embedded tumor samples from 51 patients with histologically proven colorectal adenocarcinoma referred to Madinah center in Saudi Arabia were analyzed for the occurrence of rat sarcoma virus (RAS) mutations. Results RAS mutations occurred in 43% of the patients; 91% of these mutations were in K-ras. Seventy-five percent of these K-ras mutations were in codon 12, most commonly p.G12D. Codon 13 mutations occurred in 20% of tumors: all of these were p.G13D (100%). The percentage of K-ras mutations occurrence was higher in young patients (≤50) compared with the older patients (>50) (54.5% and 35%, respectively). Similarly, the percentage of K-ras mutations occurrence was higher in the right-sided tumors compared with the left-sided tumors (57.1% and 32.4%, respectively). Patients' characteristics and clinicopathological features were not significantly associated with K-ras mutations. Conclusions K-ras mutations are common among Saudi patients diagnosed with CRC in Madinah, especially pG12V and pG12D in codon 12. Further investigation would be required to establish correlation of K-ras mutations in larger cohorts.

Failure to replicate the association of rare loss-of-function variants in type I IFN immunity genes with severe COVID-19.

A recent report found that rare predicted loss-of-function (pLOF) variants across 13 candidate genes in TLR3- and IRF7-dependent type I IFN pathways explain up to 3.5% of severe COVID-19 cases. We performed whole-exome or whole-genome sequencing of 1,934 COVID-19 cases (713 with severe and 1,221 with mild disease) and 15,251 ancestry-matched population controls across four independent COVID-19 biobanks. We then tested if rare pLOF variants in these 13 genes were associated with severe COVID-19. We identified only one rare pLOF mutation across these genes amongst 713 cases with severe COVID-19 and observed no enrichment of pLOFs in severe cases compared to population controls or mild COVID-19 cases. We find no evidence of association of rare loss-of-function variants in the proposed 13 candidate genes with severe COVID-19 outcomes.

Hypoxia Induces the Acquisition of Cancer Stem-like Phenotype Via Upregulation and Activation of Signal Transducer and Activator of Transcription-3 (STAT3) in MDA-MB-231, a Triple Negative Breast Cancer Cell Line.

The finding that hypoxia can induce cancer stemness in various experimental models is in agreement with the conceptual basis of cancer cell plasticity. Here, we aimed to gain insights into the molecular basis of hypoxia-induced cancer cell plasticity in triple negative breast cancer (TNBC). To achieve this goal, we employed our previously published in-vitro model of TNBC, in which a small subset of stem-like cells can be distinguished from the bulk cell population based on their responsiveness to a Sox2 reporter. In MDA-MB-231, a TNBC cell line, we observed that hypoxia significantly increased the expression of luciferase and green fluorescence protein (GFP), the readouts of the Sox2 reporter. Upon hypoxic challenge, the bulk, reporter unresponsive (RU) cells acquired stem-like features, as evidenced by the significant increases in the proportion of CD44high/CD24low cells, colony formation and resistance to cisplatin. Correlating with these phenotypic changes, RU cells exposed to hypoxia exhibited a substantial upregulation of the active/phosphorylated form of STAT3 (pSTAT3). This hypoxia-induced activation of STAT3 correlated with increased STAT3 transcriptional activity, as evidenced by increased STAT3-DNA binding and an altered gene expression profile. This hypoxia-induced STAT3 activation is biologically significant, since siRNA knockdown of STAT3 in RU cells significantly attenuated the hypoxia-induced acquisition of Sox2 activity and stem-like phenotypic features. In conclusion, our data have provided the proof-of-concept that STAT3 is a critical mediator in promoting the hypoxia-induced acquisition of cancer stemness in TNBC. Targeting STAT3 in TNBC may be useful in overcoming chemoresistance and decreasing the risk of disease relapse.

The absence of a novel intron 19-retaining ALK transcript (ALK-I19) and MYCN amplification correlates with an excellent clinical outcome in neuroblastoma patients.

ALK missense mutations are detected in 8% of neuroblastoma (NB) tumors at diagnosis and confer gain-of-function oncogenic effects. The mechanisms by which the expression of wild-type or mutant ALK, which is detectable in the majority of cases, is regulated are not well understood. We have identified a novel ALK transcript characterized by the retention of intron 19 (ALK-I19). ALK-I19 was detected in 4/4 NB cell lines, but not other non-NB cells with ALK aberrations. The functional significance of ALK-I19 was determined by specific siRNA knockdown of this transcript, which resulted in substantially decreased expression of the fully-spliced ALK transcripts (FS-ALK) and a significant reduction in cell growth. We also demonstrate that ALK-I19 is a precursor of FS-ALK. ALK-I19 was detected in 14/37 (38%) tumors from patients with newly diagnosed NB. ALK-I19 expression correlated with undifferentiated histology and strong ALK protein expression detectable by immunohistochemistry. Importantly, patients with tumors that did not express ALK-I19 and lacked MYCN amplification had an excellent clinical outcome, with 19/19 patients survived at 5-years. In conclusion, ALK-I19 is a novel ALK transcript that likely represents a marker of undifferentiated NB cells. The absence of ALK-I19 and MYCN amplification is a useful prognostic marker for NB patients.

Phosphorylation of Sox2 at Threonine 116 is a Potential Marker to Identify a Subset of Breast Cancer Cells with High Tumorigenecity and Stem-Like Features.

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.

Constitutive Activation of STAT3 in Myeloma Cells Cultured in a Three-Dimensional, Reconstructed Bone Marrow Model.

Malignant cells cultured in three-dimensional (3D) models have been found to be phenotypically and biochemically different from their counterparts cultured conventionally. Since most of these studies employed solid tumor types, how 3D culture affects multiple myeloma (MM) cells is not well understood. Here, we compared MM cells (U266 and RPMI8226) in a 3D culture model with those in conventional culture. While the conventionally cultured cells were present in single cells or small clusters, MM-3D cells grew in large spheroids. We discovered that STAT3 was the pathway that was more activated in 3D in both cell lines. The active form of STAT3 (phospho-STAT3 or pSTAT3), which was absent in MM cells cultured conventionally, became detectable after 1⁻2 days in 3D culture. This elevated pSTAT3 level was dependent on the 3D environment, since it disappeared after transferring to conventional culture. STAT3 inhibition using a pharmacological agent, Stattic, significantly decreased the cell viability of MM cells and sensitized them to bortezomib in 3D culture. Using an oligonucleotide array, we found that 3D culture significantly increased the expression of several known STAT3 downstream genes implicated in oncogenesis. Since most primary MM tumors are naturally STAT3-active, studies of MM in 3D culture can generate results that are more representative of the disease.

Novel Molecular Challenges in Targeting Anaplastic Lymphoma Kinase in ALK-Expressing Human Cancers.

Targeting anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase receptor initially identified as a potent oncogenic driver in anaplastic large-cell lymphoma (ALCL) in the form of nucleophosmin (NPM)-ALK fusion protein, using tyrosine kinase inhibitors has shown to be a promising therapeutic approach for ALK-expressing tumors. However, clinical resistance to ALK inhibitors invariably occurs, and the molecular mechanisms are incompletely understood. Recent studies have clearly shown that clinical resistance to ALK inhibitors is a multifactorial and complex mechanism. While few of the mechanisms of clinical resistance to ALK inhibitors such as gene mutation are well known, there are others that are not well covered. In this review, the molecular mechanisms of cancer stem cells in mediating resistance to ALK inhibitors as well as the current understanding of the molecular challenges in targeting ALK in ALK-expressing human cancers will be discussed.

High expression of ß-catenin contributes to the crizotinib resistant phenotype in the stem-like cell population in neuroblastoma.

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 ß-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 ß-catenin expression, since siRNA knockdown of ß-catenin restored the crizotinib-ALK binding and lowered the crizotinib resistance to the level of RU cells. Enforced expression of ß-catenin in RU cells resulted in the opposite effects. To conclude, high expression of ß-catenin in the stem-like NB cells contributes to their crizotinib resistance. Combining ß-catenin inhibitors and ALK inhibitors may be useful in treating NB patients.

High Myc expression and transcription activity underlies intra-tumoral heterogeneity in triple-negative breast cancer.

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.

Micellar nano-carriers for the delivery of STAT3 dimerization inhibitors to melanoma.

The objective of this research was to develop polymeric micellar formulations of inhibitors of signal transducer and activator of transcription 3 (STAT3) dimerization, i.e., S3I-1757 and S3I-201, and evaluate the activity of successful formulations in B16-F10 melanoma, a STAT3 hyperactive cancer model, in vitro and in vivo. STAT3 inhibitory agents were encapsulated in methoxy poly(ethylene oxide)-b-poly(ε-caprolactone) (PEO114-b-PCL22) and methoxy poly(ethylene oxide)-b-poly(α-benzyl carboxylate-ε-caprolactone) (PEO114-b-PBCL20) micelles using co-solvent evaporation. Polymeric micelles of S3I-1757 showed high encapsulation efficiency (>88%), slow release profile (<32% release in 24 h) under physiological conditions, and a desirable average diameter for tumor targeting (33-54 nm). The same formulations showed low encapsulation efficiencies and rapid drug release for S3I-201. Further studies evidenced the delivery of functional S3I-1757 by polymeric micelles to B16-F10 melanoma cells, leading to a dose-dependent inhibition of cell growth and vascular endothelial growth factor (VEGF) production comparable with that of free drug. Encapsulation of S3I-1757 in polymeric micelles significantly reduced its cytotoxicity in normal bone marrow-derived dendritic cells (DCs). Micelles of S3I-1757 were able to significantly improve the function of B16-F10 tumor-exposed immunosuppressed DCs in the production of IL-12, an indication for functionality in the induction of cell-mediated immune response. In a B16-F10 melanoma mouse model, S3I-1757 micelles inhibited tumor growth and enhanced the survival of tumor-bearing mice more than free S3I-1757. Our findings show that both PCL- and PBCL-based polymeric micelles have potential for the solubilization and delivery of S3I-1757, a potent STAT3 inhibitory agent.

A positive feedback loop involving the Wnt/ß-catenin/MYC/Sox2 axis defines a highly tumorigenic cell subpopulation in ALK-positive anaplastic large cell lymphoma.

BACKGROUND: We have previously described the existence of two phenotypically distinct cell subsets in ALK-positive anaplastic large cell lymphoma (ALK + ALCL) based on their differential responsiveness to a Sox2 reporter (SRR2), with reporter-responsive (RR) cells being more tumorigenic and chemoresistant than reporter-unresponsive (RU) cells. However, the regulator(s) of RU/RR dichotomy are not identified. In this study, we aim to delineate the key regulator(s) of RU/RR dichotomy. METHODS: JASPER motif match analysis was used to identify the putative factors binding to SRR2 sequence. SRR2 probe pull-down assay and quantitate real-time PCR were performed to analyze the regulation of Sox2 transcriptional activity by MYC. Methylcellulose colony formation assay, chemoresistance to doxorubicin and mouse xenograft study were performed to investigate the biological functions of MYC. PCR array and western blotting were executed to study related signaling pathways that regulate MYC expression. Immunofluorescence and immunohistochemistry assay were initiated to evaluate the expression of MYC and its correlation with its regulator by chi-square test analysis in human primary tumor cells. RESULTS: We identified MYC as a potential regulator of RU/RR dichotomy. In support of its role, MYC was highly expressed in RR cells compared to RU cells, and inhibition of MYC substantially decreased the Sox2/SRR2 binding, Sox2 transcriptional activity, chemoresistance, and methylcellulose colony formation. In contrast, enforced expression of MYC in RU cells conferred the RR phenotype. The Wnt/ß-catenin pathway, a positive regulator of MYC, was highly active in RR but not RU cells. While inhibition of this pathway in RR cells substantially decreased MYC expression and SRR2 reporter activity, experimental activation of this pathway led to the opposite effects in RU cells. Collectively, our results support a model in which a positive feedback loop involving Wnt/ß-catenin/MYC and Sox2 contributes to the RR phenotype. In a mouse xenograft model, RU cells stably transfected with MYC showed upregulation of the Wnt/ß-catenin/MYC/Sox2 axis and increased tumorigenecity. Correlating with these findings, there was a significant correlation between the expression of active ß-catenin and MYC in ALK + ALCL primary tumor cells. CONCLUSIONS: A positive feedback loop involving the Wnt/ß-catenin/MYC/Sox2 axis defines a highly tumorigenic cell subset in ALK + ALCL.