Mitotic arrest and slippage induced by pharmacological inhibition of Polo‐like kinase 1

Exposure to drugs that interfere with microtubule dynamics block cell cycle progression at mitosis by prolonged activation of the spindle assembly checkpoint (SAC). Cells can evade mitotic arrest and proceed to interphase without chromosome segregation by a process termed mitotic slippage that involves Cyclin B1 degradation without checkpoint inactivation. Here, we explored the cellular response to small‐molecule inhibitors of Polo‐like kinase 1 (Plk1), an important regulator of cell division. We found that the clinical Plk1 inhibitors BI 2536 and BI 6727, both unexpectedly, induced a dose‐dependent cellular drug response: While mitotic arrest was induced in cancer cell lines and primary non‐transformed cells across the entire range of concentrations tested, only high concentrations seemed to promote mitotic slippage. Since this observation contrasts with the effects expected from studies reporting RNAi‐mediated Plk1 depletion in cancer cells, we wondered whether both ATP‐competitive inhibitors target unknown kinases that are involved in signaling from the spindle assembly checkpoint (SAC) and might contribute to the mitotic slippage. A chemical proteomics approach used to profile the selectivity of both inhibitors revealed that SAC kinases are not targeted directly. Still, the activities of Cdk1/Cyclin B1 and Aurora B, which plays important roles in the error correction of false microtubule‐kinetochore attachments and in checkpoint signaling, were shown to be downregulated at high inhibitor concentrations. Our data suggest that the inhibition of Plk1 activity below a certain threshold influences Aurora B activity via reduced phosphorylation of Fox M1 and Survivin leading to diminished levels of Aurora B protein and alteration of its subcellular localization. Within the spectrum of SAC proteins that are degraded during mitotic slippage, the degradation of Cyclin B1 and the downregulation of Aurora B activity by Plk1 inhibition seem to be critical promoters of mitotic slippage. The results indicate that careful dose‐finding studies in cancer trials are necessary to limit or even prevent mitotic slippage, which could be associated with improved cancer cell survival.     

miR‐203 induces oxaliplatin resistance in colorectal cancer cells by negatively regulating ATM kinase

Chemotherapy for patients with metastatic colorectal cancer (CRC) is the standard of care, but ultimately nearly all patients develop drug resistance. Understanding the mechanisms that lead to resistance to individual chemotherapeutic agents may help identify novel targets and drugs that will, in turn, improve therapy. Oxaliplatin is a common component combination therapeutic regimen for use in patients with metastatic CRC, but is also used as a component of adjuvant therapy for patients at risk for recurrent disease. In this study, unbiased microRNA array screening revealed that the miR‐203 microRNA is up‐regulated in three of three oxaliplatin‐resistant CRC cell lines, and therefore we investigated the role of miR‐203 in chemoresistance. Exogenous expression of miR‐203 in chemo‐naïve CRC cells induced oxaliplatin resistance. Knockdown of miR‐203 sensitized chemoresistant CRC cells to oxaliplatin. In silico analysis identified ataxia telangiectasia mutated (ATM), a primary mediator of the DNA damage response, as a potential target of miR‐203. ATM mRNA and protein levels were significantly down‐regulated in CRC cells with acquired resistance to oxaliplatin. Using TCGA database, we identified a significant reverse correlation of miR‐203 and ATM expression in CRC tissues. We validated ATM as a bona fide target of miR‐203 in CRC cells. Mutation of the putative miR‐203 binding site in the 3′ untranslated region (3′UTR) of the ATM mRNA abolished the inhibitory effect of miR‐203 on ATM. Furthermore, stable knockdown of ATM induced resistance to oxaliplatin in chemo‐naïve CRC cells. This is the first report of oxaliplatin resistance in CRC cells induced by miR‐203‐mediated suppression of ATM.     

Spontaneous transformation of human adult non-tumorigenic stem cells to cancer stem cells is driven by genomic instability in a human model of glioblastoma

The presence of a CD133＋/Nestin＋ population in brain tumors suggests that a normal neural stem cell （NSC） may be the cell of origin for gliomas. We have identified human CD133 positive NSCs from adult ghoma tissue and established them as long term in vitro cultures HNGC-1 （Human Neuro Glial Culture）. Replicative senescence in HNGC-1 led to high level of genomie instability and emergence of a spontaneously immortalized clone that developed into a cell-line HNC_,C-2 with features of cancer stem cells （CSCs） that includes ability for serf-renewal,     

Wnt/β-catenin signaling is a key downstream mediator of MET signaling in glioblastoma stem cells

Background

Glioblastoma (GBM) is the most lethal and common type of primary brain tumor. Recent evidence suggests that a subpopulation of GBM cells (glioblastoma stem cells [GSCs]) is critical for tumor progression, invasion, and therapeutic resistance. We and others have demonstrated that MET, a receptor tyrosine kinase, positively regulates the stemness phenotype and radioresistance of GSCs. Here, we interrogated the downstream effector pathways of MET signaling in GSCs.

Methods

We have established a series of GSCs and xenograft tumors derived from freshly dissociated specimens from patients with GBM and characterized a subpopulation enriched with MET activation (MET^high/+). Through global expression profiling and subsequent pathways analysis, we identified signaling pathways that are enriched in MET^high/+ populations, one of which is Wnt/β-catenin signaling pathway. To determine molecular interaction and the biological consequences of MET and Wnt/β-catenin signaling, we used pharmacological and shRNA-mediated genetic inhibition and performed various molecular and cellular analyses, including flow cytometry, immunohistochemistry, and clonogenicity assays.

Results

We found that Wnt/β-catenin signaling is highly active in MET^high/+ cells, compared with bulk tumor cells. We also showed that Wnt/β-catenin signaling activities in GBM are directly modulated by the addition of ligand-mediated MET activation or MET inhibition. Furthermore, the ectopic expression of active-β-catenin (S37A and S45Y) rescued the phenotypic effects caused by MET inhibition.

Conclusion

These data suggest that Wnt/β-catenin signaling is a key downstream effector of MET signaling and contributes to the maintenance of GSC and GBM malignancy.     

Sensitization of tumor cells to cancer therapy by molecularly targeted inhibition of the inhibitor of nuclear factor κB kinase

The inhibitor of nuclear factor κB kinase (IKK)-nuclear factor κB (NFκB) pathway is one of the most important cellular signal transduction pathways. It can be activated by diverse stimuli, resulting in liberation of cytoplasmic NFκB from inhibition by inhibitors of NFκB (IκB) after IκB are phosphorylated by IKKβ and IKKα via the canonical and non-canonical pathways, respectively. Activated NFκB then translocates into the nucleus to regulate various NFκB target genes. Through regulation of its target genes, NFκB can regulate various physiologic processes such as cell proliferation, migration and survival. More importantly, activation of the IKK-NFκB pathway has been implicated in carcinogenesis, tumor development, progression and metastasis, and cancer resistance to radiotherapy and chemotherapy. Therefore, molecularly targeted inhibition of the different components of this pathway has been widely explored for treatment of cancer either alone or in combination with other cancer therapies. A growing body of evidence suggests that IKKβ may be a better cancer treatment target in this pathway, because several novel NFκB-independent functions of IKKβ have been identified recently, including promotion of DNA double strand break repair to increase tumor cell resistance to ionizing radiation and chemotherapy in an apoptosis-independent manner. In this review, we highlight some of these new findings and discuss the therapeutic potential of IKKβ specific inhibitors as a novel tumor sensitizer.     

HIF-1α inhibition by siRNA or chetomin in human malignant glioma cells: effects on hypoxic radioresistance and monitoring via CA9 expression

Background  

Hypoxia induces activation of the HIF-1 pathway and is an essential characteristic of malignant gliomas. Hypoxia has been linked to tumor progression, therapy resistance and poor prognosis. However, little is known about the impact of HIF-1α inhibition on radioresistance of malignant glioma.     

Regulation of BAX and BCL‐2 expression in breast cancer cells by chemotherapy

Optimizing chemotherapeutic drug delivery strategies relies, in part, on identification of the most clinically effective sequence, dose, and duration of drug exposure. The combination of dose intensive etoposide (VP16) followed by cyclophosphamide has clinical efficacy in the treatment of advanced breast cancer. However, molecular mechanisms that underlie the effectiveness of this combination of chemotherapeutic agents have not been investigated. In this study we investigated regulation of BAX and BCL2 expression by VP16 and cyclophosphamide as a potential mechanism for the induction of breast cancer cell death induced by this regimen.There was a dose and time dependent increase in BAX expression in the breast cancer cell lines MCF7, MDAMB435S, and MDAMBA231 following in vitro treatment with 50–100M VP16. Elevation of BAX protein expression in the presence of VP16 alone did not correlate with reduced viability or induction of apoptosis in MCF7, MDAMB435S, or MDAMBA231. VP16 did effectively block the breast cancer cell lines evaluated (MCF7 and MDAMB435S) at G₂/M phase of the cell cycle, confirming activity of the drug in vitro. MCF7 and MDAMB435S cells that were pretreated with VP16 and subsequently exposed to 1.0–12.0g/m1 4hydroperoxycyclophosphamide (4HC), an active metabolite of cyclophosphamide, had markedly reduced viability when compared to matched controls treated with either VP16 or 4HC individually. Consistent with this loss of viability, exposure of all three cell lines to the combination of VP16 and 4HC resulted in higher BAX protein levels than those observed following treatment with either single agent. This combination of chemotherapeutic agents also resulted in reduced BCL2 expression.These observations suggest that combination chemotherapy may derive its efficacy, in part, through coordinated regulation of specific gene products associated with apoptosis. Characterization of molecular events that underlie susceptibility of specific tumor cells to combination chemotherapeutic regimens may lead to additional improvements in treatment strategies for this disease.     

MiR‐449a exerts tumor‐suppressive functions in human glioblastoma by targeting Myc‐associated zinc‐finger protein

Glioblastoma (GBM) is one of the most common and aggressive primary brain tumors in adults. Deregulated expression of microRNAs (miRNAs) has been associated with GBM progression through alterations in either oncogenic or tumor suppressor targets. Here, we elucidated the function and the possible molecular mechanisms of miR‐449a in human GBM cell lines and tumor specimens‐derived glioblastoma stem cells (GSCs). Quantitative real‐time PCR demonstrated that miR‐449a was down‐regulated in human GBM cell lines and GSCs. Functionally, miR‐449a acted as a tumor suppressor by reducing cell proliferation, migration and invasion as well as inducing apoptosis in human GBM cell lines and GSCs. Myc‐associated zinc‐finger protein (MAZ) was identified as a direct target of miR‐449a, mediating these tumor‐suppressive effects, demonstrated by Western blot assay and luciferase assays. Moreover, over‐expression of miR‐449a inhibited the expression of Podoplanin (PDPN) by down‐regulating MAZ which could positively control the promoter activities via binding to the promoter of PDPN, demonstrated by luciferase assays and chromatin immunoprecipitation assays. Further, the PI3K/AKT pathway was blocked when MAZ was down‐regulated by miR‐449a. This process was coincided with the up‐regulation of apoptotic proteins and the down‐regulation of anti‐apoptotic proteins, MMP2 and MMP9. Furthermore, nude mice carrying over‐expressed miR‐449a combined with knockdown MAZ tumors produced the smallest tumors and the highest survival. These results elucidated a novel molecular mechanism of GBM progression, and may thus suggest a promising application for GBM treatment.     

Are genetic determinants of asymmetric stem cell division active in hematopoietic stem cells?

Stem cells have acquired a golden glow in the past few years as they represent possible tools for reversing the damage wreak on organs. These cells are found not only in major regenerative tissues, such as the epithelia, blood and testes, but also in 'static tissues', such as the nervous system and liver, where they play a central role in tissue growth and maintenance. The mechanism by which stem cells maintain populations of highly differentiated, short-lived cells seems to involve a critical balance between alternate fates: daughter cells either maintain stem cell identity or initiate differentiation. Recent studies in lower organisms have unveiled the regulatory mechanisms of asymmetric stem cell divisions. In these models, the surrounding environment likely provides key instructive signals for the cells to choose one fate over another. Our understanding now extends to the intrinsic mechanisms of cell polarity that influence asymmetrical stem cell divisions. This article focuses on the genetic determinants of asymmetric stem cell divisions in lower organisms as a model for studying the process of self-renewal of mammalian hematopoietic stem cells.     

MiR-196a exerts its oncogenic effect in glioblastoma multiforme by inhibition of IκBα both in vitro and in vivo

Background

Recent studies have revealed that miR-196a is upregulated in glioblastoma multiforme (GBM) and that it correlates with the clinical outcome of patients with GBM. However, its potential regulatory mechanisms in GBM have never been reported.

Methods

We used quantitative real-time PCR to assess miR-196a expression levels in 132 GBM specimens in a single institution. Oncogenic capability of miR-196a was detected by apoptosis and proliferation assays in U87MG and T98G cells. Immunohistochemistry was used to determine the expression of IκBα in GBM tissues, and a luciferase reporter assay was carried out to confirm whether IκBα is a direct target of miR-196a. In vivo, xenograft tumors were examined for an antiglioma effect of miR-196a inhibitors.

Results

We present for the first time evidence that miR-196a could directly interact with IκBα 3′-UTR to suppress IκBα expression and subsequently promote activation of NF-κB, consequently promoting proliferation of and suppressing apoptosis in GBM cells both in vitro and in vivo. Our study confirmed that miR-196a was upregulated in GBM specimens and that high levels of miR-196a were significantly correlated with poor outcome in a large cohort of GBM patients. Our data from human tumor xenografts in nude mice treated with miR-196 inhibitors demonstrated that inhibition of miR-196a could ameliorate tumor growth in vivo.

Conclusions

MiR-196a exerts its oncogenic effect in GBM by inhibiting IκBα both in vitro and in vivo. Our findings provide new insights into the pathogenesis of GBM and indicate that miR-196a may predict clinical outcome of GBM patients and serve as a new therapeutic target for GBM.     

Generation of reactive oxygen species by xanthine derivatives in MDA‐MB‐231 human breast cancer cells

Theophylline reduces cell number in MDAMB231 cells through mechanisms over and above phosphodiesterase inhibition. In the current study, we used an intracellular fluorescent dye to show that theophylline and, to a much greater extent, 3isobutyl1methylxanthine, evoke the generation of reactive oxygen species and also sensitize the cells to insult by other oxidants. Xanthine derivatives may therefore offer novel strategies for antitumor therapeutics.     

Temozolomide decreases invasion of glioma stem cells by down-regulating TGF-β2

Gliomas are characterized by excessive proliferation, diffuse infiltration and immunosuppression. Recent studies implicate a key role for a restricted population of glioma stem cells (GSCs) in glioma invasive growth and recurrence. Transforming growth factor (TGF)-β2 is a mediator of immunosuppression associated with malignant glioma and also influences pro-invasive functions. Temozolomide (TMZ), is a new alkylating agent with promising antitumour efficacy for malignant gliomas, and the effect of TMZ on GSCs invasion has not been known. To address this issue, we developed studies aimed at neurospheres from primary cultured glioma cells, due to the fact that since neurospheres can be enriched in GSCs, we could examine whether TMZ inhibits the invasion of GSCs. TMZ reduced the TGF-β2-mediated invasion, and down-regulated TGF-β2 expression at the mRNA and protein levels. Thus, these results indicate that TMZ, as a chemotherapeutic agent, can reduce the invasion of GSCs and their immunosuppressive activity. TMZ may be used as an immunomodulating agent for glioma therapy.     

Targeting of VEGF‐dependent transendothelial migration of cancer cells by bevacizumab

Cancer progression is often associated with the formation of malignant effusions. Vascular endothelial growth factor (VEGF) is a major regulator of vascular permeability and has been implicated as mediator of tumor progression.We examined the production and secretion of VEGF(165) in various primary cancer cells derived from malignant effusions, and the role of exogenous VEGF165 as a mediator of effusion formation. VEGF165 was constantly secreted by all cultured tumor cells in an mTOR‐dependent manner, as it was inhibited by the mTOR inhibitor rapamycin. Secreted VEGF165 showed functional activity by inducing endothelial leakiness and tumor cell‐transendothelial migration in vitro, effects which could be reverted by the anti‐VEGF antibody bevacizumab.Thus, mTOR inhibitors as well as bevacizumab should be considered as potential agents in cancer patients suffering from malignant effusions.     

Radioresistance of glioma stem cells: Intrinsic characteristic or property of the ‘microenvironment‐stem cell unit’?

There is increasing evidence that glioblastoma possess 'stem-like' cells, low concentrations of which can initiate a tumour. It has been proposed that these cells are radioresistant, and that this property contributes to the poor treatment outcomes of these tumours. In this paper we propose that radioresistance is not simply an intrinsic characteristic of glioma stem cells but a result of interactions between these cells and microenvironmental factors, i.e. the 'microenvironment - stem cell unit'. The critical role of the microenvironment, along with glioma stem cells, is supported directly or indirectly by the following observations: glioma stem cells have been shown to reside preferentially in specific niches, the characteristics of which are known to influence cellular responses to radiation; radiation modifies environmental factors; and, contrarily to the consistency of clinical data, in vitro experiments have reported a wide variety in the radiation response of these cells. The paper, therefore, focuses on the interaction between tumour stem cells and the microenvironment, analyzing how its various elements (endothelial cells, extracellular matrix, cytokines, nitric oxide, oxygen levels) are affected by radiation and how these might influence the response of tumour stem cells to radiation. Finally, we summarize the ongoing debate on the optimal culture conditions for glioma stem cells and the difficulties in designing assays that reliably characterize their radiation response.