Self-renewal of acute lymphocytic leukemia cells is limited by the Hedgehog pathway inhibitors cyclopamine and IPI-926

Conserved embryonic signaling pathways such as Hedgehog (Hh), Wingless and Notch have been implicated in the pathogenesis of several malignancies. Recent data suggests that Hh signaling plays a role in normal B-cell development, and we hypothesized that Hh signaling may be important in precursor B-cell acute lymphocytic leukemia (B-ALL). We found that the expression of Hh pathway components was common in human B-ALL cell lines and clinical samples. Moreover, pathway activity could be modulated by Hh ligand or several pathway inhibitors including cyclopamine and the novel SMOOTHENED (SMO) inhibitor IPI-926. The inhibition of pathway activity primarily impacted highly clonogenic B-ALL cells expressing aldehyde dehydrogenase (ALDH) by limiting their self-renewal potential both in vitro and in vivo. These data demonstrate that Hh pathway activation is common in B-ALL and represents a novel therapeutic target regulating self-renewal and persistence of the malignant clone.     

Autocrine-mediated activation of STAT3 correlates with cell proliferation in breast carcinoma lines

The intracellular signals driving the proliferation of breast carcinoma (BC) cells have been widely studied. Both the mitotic and metastatic potential of BC cells have been linked to the frequent overexpression of ErbB family members. Other signaling molecules, including the estrogen receptor, the tyrosine kinases c-Src and Syk, and STAT proteins, especially STAT3, have also been implicated in BC tumor growth. Here we have examined ErbB and STAT protein expression and activation in six BC-derived cell lines. ErbB expression and tyrosine phosphorylation varied considerably among the six cell lines. However, STAT protein expression and activation were more consistent. Two levels of STAT3 activation were distinguished in DNA-binding assays: an epidermal growth factor-inducible, high level that requires both ErbB1 and Janus kinase (JAK) activity and an elevated serum-dependent level that is maintained by autocrine/paracrine signaling and requires JAK activity but is independent of ErbB1 kinase activity. BC cell growth could be inhibited by dominant-negative versions of STAT3 and the JAK inhibitor AG490 but not by PD153035 or PD168393, inhibitors of ErbB1 kinase activity. This indicates that BC cell proliferation may be a consequence of STAT3 activation by autocrine/paracrine signals.     

STAT3 is activated by JAK2 independent of key oncogenic driver mutations in non-small cell lung carcinoma

Constitutive activation of STAT3 is a common feature in many solid tumors including non-small cell lung carcinoma (NSCLC). While activation of STAT3 is commonly achieved by somatic mutations to JAK2 in hematologic malignancies, similar mutations are not often found in solid tumors. Previous work has instead suggested that STAT3 activation in solid tumors is more commonly induced by hyperactive growth factor receptors or autocrine cytokine signaling. The interplay between STAT3 activation and other well-characterized oncogenic "driver" mutations in NSCLC has not been fully characterized, though constitutive STAT3 activation has been proposed to play an important role in resistance to various small-molecule therapies that target these oncogenes. In this study we demonstrate that STAT3 is constitutively activated in human NSCLC samples and in a variety of NSCLC lines independent of activating KRAS or tyrosine kinase mutations. We further show that genetic or pharmacologic inhibition of the gp130/JAK2 signaling pathway disrupts activation of STAT3. Interestingly, treatment of NSCLC cells with the JAK1/2 inhibitor ruxolitinib has no effect on cell proliferation and viability in two-dimensional culture, but inhibits growth in soft agar and xenograft assays. These data demonstrate that JAK2/STAT3 signaling operates independent of known driver mutations in NSCLC and plays critical roles in tumor cell behavior that may not be effectively inhibited by drugs that selectively target these driver mutations.     

MicroRNA-363-mediated downregulation of S1PR1 suppresses the proliferation of hepatocellular carcinoma cells

S1PR1 plays a crucial role in promoting proliferation of hepatocellular carcinoma (HCC). Over expression of S1PR1 is observed in HCC cell lines. The mechanisms underlying the aberrant expression of S1PR1 are not known well. MircroRNAs are important regulators of gene expression and disproportionate microRNAs can result in dysregulation of oncogenes in cancer cells. In this study, we found that miR-363, a potential tumor suppressor microRNA, downregulated the expression of S1PR1 and inhibited the proliferation of HCC cells. Bioinformatic analysis predicted a putative binding site of miR-363 within the 3′-UTR of S1PR1 mRNA. Luciferase reporter assay showed that miR-363 directly targeted the 3′-UTR of S1PR1 mRNA. Transfection of miR-363 mimics suppressed S1PR1 expression in HCC cells, followed by the repression of the activation of ERK and STAT3. Moreover, we found that the expression of downstream genes of ERK and STAT3, including PDGF-A, PDGF-B, MCL-1 and Bcl-xL, was suppressed after miR-363 transfection. Taken together, the present study demonstrated that miR-363 was a negative regulator of S1PR1 expression in HCC cells and inhibited cell proliferation, suggesting that the miR-363/S1PR1 pathway might be a novel target for the treatment of HCC.     

IL-6-induced Bcl6 variant 2 supports IL-6-dependent myeloma cell proliferation and survival through STAT3

IL-6 is a growth and survival factor for myeloma cells, although the mechanism by which it induces myeloma cell proliferation through gene expression is largely unknown. Microarray analysis showed that some B-cell lymphoma-associated oncogenes such as Bcl6, which is absent in normal plasma cells, were upregulated by IL-6 in IL-6-dependent myeloma cell lines. We found that Bcl6 variant 2 was upregulated by STAT3. ChIP assay and EMSA showed that STAT3 bound to the upstream region of variant 2 DNA. Expression of p53, a direct target gene of Bcl6, was downregulated in the IL-6-stimulated cells, and this process was impaired by an HDAC inhibitor. Bcl6 was knocked down by introducing small hairpin RNA, resulting in decreased proliferation and increased sensitivity to a DNA damaging agent. Thus, STAT3-inducible Bcl6 variant 2 appears to generate an important IL-6 signal that supports proliferation and survival of IL-6-dependent myeloma cells.     

Long non-coding RNA SNHG1 suppresses cell migration and invasion and upregulates SOCS2 in human gastric carcinoma

Gastric carcinoma (GC) is one of the most common malignancies and the third leading cause of cancer-related deaths worldwide. Long noncoding RNAs (lncRNAs) may be an important class of functional regulators involved in human gastric cancers development. In this study, we investigated the clinical significance and function of lncRNA SNHG1 in GC. SNHG1 was significantly downregulated in GC tumor tissues compared with adjacent noncancerous tissues. Overexpression of SNHG1 in BGC-823 cells remarkably inhibited not only cell proliferation, migration, invasion in vitro, but also tumorigenesis and lung metastasis in the chick embryo chorioallantoic membrane (CAM) assay in vivo. Conversely, inhibition of SNHG1 by transfection of siRNA in AGS cells resulted in opposite phenotype changes. Mechanically, SNHG1 was found interacted with ILF3, NONO and SFPQ. RNA-seq combined with bioinformatic analysis identified a serial of downstream genes of SNHG1, including SOCS2, LOXL2, LTBP3, LTBP4. Overexpression of SNHG1 induced SOCS2 expression whereas knockdown of SNHG1 decreased SOCS2 expression. In addition, knockdown of SNHG1 promoted the activation of JAK2/STAT signaling pathway. Taken together, our data suggested that SNHG1 suppressed aggressive phenotype of GC cells and regulated SOCS2/JAK2/STAT pathway.     

Constitutive Activation of Signal Transducer and Activator of Transcription 3 (STAT3) and Nuclear Factor κB Signaling in Glioblastoma Cancer Stem Cells Regulates the Notch Pathway

Malignant gliomas are locally aggressive, highly vascular tumors that have a dismal prognosis, and present therapies provide little improvement in the disease course and outcome. Many types of malignancies, including glioblastoma, originate from a population of cancer stem cells (CSCs) that are able to initiate and maintain tumors. Although CSCs only represent a small fraction of cells within a tumor, their high tumor-initiating capacity and therapeutic resistance drives tumorigenesis. Therefore, it is imperative to identify pathways associated with CSCs to devise strategies to selectively target them. In this study, we describe a novel relationship between glioblastoma CSCs and the Notch pathway, which involves the constitutive activation of STAT3 and NF-κB signaling. Glioma CSCs were isolated and maintained in vitro using an adherent culture system, and the biological properties were compared with the traditional cultures of CSCs grown as multicellular spheres under nonadherent culture conditions. Interestingly, both adherent and spheroid glioma CSCs show constitutive activation of the STAT3/NF-κB signaling pathway and up-regulation of STAT3- and NF-κB-dependent genes. Gene expression profiling also identified components of the Notch pathway as being deregulated in glioma CSCs, and the deregulated expression of these genes was sensitive to treatment with STAT3 and NF-κB inhibitors. This finding is particularly important because Notch signaling appears to play a key role in CSCs in a variety of cancers and controls cell fate determination, survival, proliferation, and the maintenance of stem cells. The constitutive activation of STAT3 and NF-κB signaling pathways that leads to the regulation of Notch pathway genes in glioma CSCs identifies novel therapeutic targets for the treatment of glioma.     

Cellular target genes of Epstein-Barr virus nuclear antigen 2

Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) is a key determinant in the EBV-driven B-cell growth transformation process. By activating an array of viral and cellular target genes, EBNA-2 initiates a cascade of events which ultimately cause cell cycle entry and the proliferation of the infected B cell. In order to identify cellular target genes that respond to EBNA-2 in the absence of other viral factors, we have performed a comprehensive search for EBNA-2 target genes in two EBV-negative B-cell lines. This screen identified 311 EBNA-2-induced and 239 EBNA-2-repressed genes that were significantly regulated in either one or both cell lines. The activation of most of these genes had not previously been attributed to EBNA-2 function and will be relevant for the identification of EBNA-2-specific contributions to EBV-associated malignancies. The diverse spectrum of EBNA-2 target genes described in this study reflects the broad spectrum of EBNA-2 functions involved in virus-host interactions, including cell signaling molecules, adapters, genes involved in cell cycle regulation, and chemokines.     

Resveratrol Induces Cell Cycle Arrest and Apoptosis in Malignant NK Cells via JAK2/STAT3 Pathway Inhibition

Natural killer (NK) cell malignancies, particularly aggressive NK cell leukaemias and lymphomas, have poor prognoses. Although recent regimens with L-asparaginase substantially improved outcomes, novel therapeutic approaches are still needed to enhance clinical response. Resveratrol, a naturally occurring polyphenol, has been extensively studied for its anti-inflammatory, cardioprotective and anti-cancer activities. In this study, we investigated the potential anti-tumour activities of resveratrol against the NK cell lines KHYG-1, NKL, NK-92 and NK-YS. Resveratrol induced robust G0/G1 cell cycle arrest, significantly suppressed cell proliferation and induced apoptosis in a dose- and time-dependent manner for all four cell lines. In addition, resveratrol suppressed constitutively active STAT3 in all the cell lines and inhibited JAK2 phosphorylation but had no effect on other upstream mediators of STAT3 activation, such as PTEN, TYK2, and JAK1. Resveratrol also induced downregulation of the anti-apoptotic proteins MCL1 and survivin, two downstream effectors of the STAT3 pathway. Finally, resveratrol induced synergistic effect on the apoptotic and antiproliferative activities of L-asparaginase against KHYG-1, NKL and NK-92 cells. These results suggest that resveratrol may have therapeutic potential against NK cell malignancies. Furthermore, our finding that resveratrol is a bonafide JAK2 inhibitor extends its potential benefits to other diseases with dysregulated JAK2 signaling.     

Overexpression of heme oxygenase-1 in microenvironment mediates vincristine resistance of B-cell acute lymphoblastic leukemia by promoting vascular endothelial growth factor secretion

Chemoresistance often causes treatment failure of B-cell acute lymphoblastic leukemia (B-ALL). However, the mechanism remains unclear at present. Herein, overexpression of heme oxygenase-1 (HO-1) was found in the bone marrow stromal cells (BMSCs) from B-ALL patients developing resistance to vincristine (VCR), a chemotherapeutic agent. Two B-ALL cell lines Super B15 and CCRF-SB were cocultured with BMSCs transfected with lentivirus to regulate the expression of HO-1. Silencing HO-1 expression in BMSCs increased the apoptotic rates of B-ALL cell lines induced by VCR, whereas upregulating HO-1 expression reduced the rate. Cell cycle can be arrested in the G2/M phase by VCR. In contrast, B-ALL cells were arrested in the G0/G1 phase due to HO-1 overexpression in BMSCs, which avoided damage from the G2/M phase. Vascular endothelial growth factor (VEGF) in BMSCs, as a key factor in the microenvironment-associated chemoresistance, was also positively coexpressed with HO-1. VEGF secretion was markedly increased in BMSCs with HO-1 upregulation but decreased in BMSCs with HO-1 silencing. B-ALL cell lines became resistant to VCR when cultured with VEGF recombinant protein, so VEGF secretion induced by HO-1 expression may promote the VCR resistance of B-ALL cells. As to the molecular mechanism, the PI3K/AKT pathway mediated regulation of VEGF by HO-1. In conclusion, this study clarifies a mechanism by which B-ALL is induced to resist VCR through HO-1 overexpression in BMSCs, and provides a novel strategy for overcoming VCR resistance in clinical practice.     

Convallatoxin promotes apoptosis and inhibits proliferation and angiogenesis through crosstalk between JAK2/STAT3 (T705) and mTOR/STAT3 (S727) signaling pathways in colorectal cancer

BackgroundAberrant activation of STAT3 is frequently encountered and promotes survival, cellular proliferation, migration, invasion and angiogenesis in tumor cell. Convallatoxin, triterpenoid ingredient, exhibits anticancer pharmacological properties.PurposeIn this work, we investigated the anticancer potential of convallatoxin and explored whether convallatoxin mediates its effect through interference with the STAT3 activation in colorectal cancer cells.MethodsIn vitro, the underlying mechanisms of convallatoxin at inhibiting STAT3 activation were investigated by homology modeling and molecular docking, luciferase reporter assay, MTT assay, RT-PCR, Western blotting and immunofluorescence assays. Changes in cellular proliferation, apoptosis, migration, invasion and angiogenesis were analyzed by EdU labeling assay, colony formation assay, flow cytometry assay, wound-healing assay, matrigel transwell invasion assay and tube formation assays. And in vivo, antitumor activity of convallatoxin was assessed in a murine xenograft model of HCT116 cells.ResultsConvallatoxin decreased the viability of colorectal cancer lines. Moreover, convallatoxin reduced the P-STAT3 (T705) via the JAK1, JAK2, and Src pathways and inhibited serine-727 phosphorylation of STAT3 via the PI3K-AKT-mTOR-STAT3 pathways in colorectal cancer cells. Interestingly, we discovered the crosstalk between mTOR and JAK2 in mTOR/STAT3 and JAK/STAT3 pathways, which collaboratively regulated STAT3 activation and convallatoxin play a role in it. Convallatoxin also downregulated the expression of target genes involved cell survival (e.g., Survivin, Bcl-xl, Bcl-2), proliferation (e.g., Cyclin D1), metastasis (e.g., MMP-9), and angiogenesis (e.g., VEGF). Indeed, we found that convallatoxin inhibited tube formation, migration, and invasion of endothelial cells, and inhibited the proliferation. Finally, in vivo observations were confirmed by showing antitumor activity of convallatoxin in a murine xenograft model.ConclusionThe result of the current study show that convallatoxin promotes apoptosis and inhibits proliferation and angiogenesis through crosstalk between JAK2/STAT3 (T705) and mTOR/STAT3 (S727) signaling pathways in colorectal cancer cells and indicate that convallatoxin could be a valuable candidate for the development of colorectal cancer therapeutic.     

Nucleotide excision repair and B lymphoma: Somatic hypermutation is not the only culprit

Different mechanisms account for the development of B lymphoma. Malignant transformation of B lymphocytes arises from progressive loss of genome integrity combined with uncontrolled cell proliferation, often triggered by foreign or self antigens. It is well established that somatic hypermutation, the pathway responsible for introducing high levels of mutations in immunoglobulin genes, also targets several other genes, contributing mainly to germinal center-derived B-cell lymphoma. We have recently discovered that a major DNA repair pathway, nucleotide excision repair (NER), is downregulated in quiescent B lymphocytes. Upon B-cell stimulation, unrepaired DNA damage results in the accumulation of mutations in a different and likely larger set of genes, including normally silent genes (e.g., oncogenes) as well as cell cycle and activation-induced genes. This mechanism potentially produces a transforming event relevant to a wider palette of B lymphomas. Here we discuss the relative contribution of both mechanisms to lymphomagenesis and possible implications of NER downregulation for other types of malignancies and for B cell-mediated immunity. Given that hematopoietic cancer stem cells remain quiescent for long periods of time, we propose that downregulation of NER during quiescence, in an environment that causes both genotoxic stress and proliferation, could be a general mechanism for carcinogenesis.