技术资料

Myelodysplastic syndromes (MDS) are characterized by abnormal and dysplastic maturation of all blood lineages. Even though epigenetic alterations have been seen in MDS marrow progenitors,very little is known about the molecular alterations in dysplastic peripheral blood cells. We analyzed the methylome of MDS leukocytes by the HELP assay and determined that it was globally distinct from age-matched controls and was characterized by numerous novel,aberrant hypermethylated marks that were located mainly outside of CpG islands and preferentially affected GTPase regulators and other cancer-related pathways. Additionally,array comparative genomic hybridization revealed that novel as well as previously characterized deletions and amplifications could also be visualized in peripheral blood leukocytes,thus potentially reducing the need for bone marrow samples for future studies. Using integrative analysis,potentially pathogenic genes silenced by genetic deletions and aberrant hypermethylation in different patients were identified. DOCK4,a GTPase regulator located in the commonly deleted 7q31 region,was identified by this unbiased approach. Significant hypermethylation and reduced expression of DOCK4 in MDS bone marrow stem cells was observed in two large independent datasets,providing further validation of our findings. Finally,DOCK4 knockdown in primary marrow CD34(+) stem cells led to decreased erythroid colony formation and increased apoptosis,thus recapitulating the bone marrow failure seen in MDS. These findings reveal widespread novel epigenetic alterations in myelodysplastic leukocytes and implicate DOCK4 as a pathogenic gene located on the 7q chromosomal region. View Publication

The phosphatidylinositol-3,4,5-triphosphate (PIP3) binding function of pleckstrin homology (PH) domain is essential for the activation of oncogenic Akt/PKB kinase. Following the PIP3-mediated activation at the membrane,the activated Akt is subjected to other regulatory events,including ubiquitination-mediated deactivation. Here,by identifying and characterizing an allosteric inhibitor,SC66,we show that the facilitated ubiquitination effectively terminates Akt signaling. Mechanistically,SC66 manifests a dual inhibitory activity that directly interferes with the PH domain binding to PIP3 and facilitates Akt ubiquitination. A known PH domain-dependent allosteric inhibitor,which stabilizes Akt,prevents the SC66-induced Akt ubiquitination. A cancer-relevant Akt1 (e17k) mutant is unstable,making it intrinsically sensitive to functional inhibition by SC66 in cellular contexts in which the PI3K inhibition has little inhibitory effect. As a result of its dual inhibitory activity,SC66 manifests a more effective growth suppression of transformed cells that contain a high level of Akt signaling,compared with other inhibitors of PIP3/Akt pathway. Finally,we show the anticancer activity of SC66 by using a soft agar assay as well as a mouse xenograft tumor model. In conclusion,in this study,we not only identify a dual-function Akt inhibitor,but also demonstrate that Akt ubiquitination could be chemically exploited to effectively facilitate its deactivation,thus identifying an avenue for pharmacological intervention in Akt signaling. View Publication

INTRODUCTION Triple-negative breast cancer (TNBC) high rate of relapse is thought to be due to the presence of tumor-initiating cells (TICs),molecularly defined as being CD44high/CD24-/low. TICs are resilient to chemotherapy and radiation. However,no currently accepted molecular target exists against TNBC and,moreover,TICs. Therefore,we sought the identification of kinase targets that inhibit TNBC growth and eliminate TICs. METHODS A genome-wide human kinase small interfering RNA (siRNA) library (691 kinases) was screened against the TNBC cell line SUM149 for growth inhibition. Selected siRNAs were then tested on four different breast cancer cell lines to confirm the spectrum of activity. Their effect on the CD44high subpopulation and sorted CD44high/CD24-/low cells of SUM149 also was studied. Further studies were focused on polo-like kinase 1 (PLK1),including its expression in breast cancer cell lines,effect on the CD44high/CD24-/low TIC subpopulation,growth inhibition,mammosphere formation,and apoptosis,as well as the activity of the PLK1 inhibitor,BI 2536. RESULTS Of the 85 kinases identified in the screen,28 of them were further silenced by siRNAs on MDA-MB-231 (TNBC),BT474-M1 (ER+/HER2+,a metastatic variant),and HR5 (ER+/HER2+,a trastuzumab-resistant model) cells and showed a broad spectrum of growth inhibition. Importantly,12 of 28 kinases also reduced the CD44high subpopulation compared with control in SUM149. Further tests of these 12 kinases directly on a sorted CD44high/CD24-/low TIC subpopulation of SUM149 cells confirmed their effect. Blocking PLK1 had the greatest growth inhibition on breast cancer cells and TICs by about 80% to 90% after 72 hours. PLK1 was universally expressed in breast cancer cell lines,representing all of the breast cancer subtypes,and was positively correlated to CD44. The PLK1 inhibitor BI 2536 showed similar effects on growth,mammosphere formation,and apoptosis as did PLK1 siRNAs. Finally,whereas paclitaxel,doxorubicin,and 5-fluorouracil enriched the CD44high/CD24-/low population compared with control in SUM149,subsequent treatment with BI 2536 killed the emergent population,suggesting that it could potentially be used to prevent relapse. CONCLUSION Inhibiting PLK1 with siRNA or BI 2536 blocked growth of TNBCs including the CD44high/CD24-/low TIC subpopulation and mammosphere formation. Thus,PLK1 could be a potential therapeutic target for the treatment of TNBC as well as other subtypes of breast cancer. View Publication

Targeted FISH analysis is an essential component of the management of plasma cell myeloma for identification of cytogenetic abnormalities. The purpose of this study was to evaluate the column-free method,RoboSep® (RS),for sorting CD138-expressing cells in bone marrow aspirates. Comparative analysis of column-based and RS methodologies was carried out on 54 paired bone marrow aspirate validation samples from patients undergoing work-up for plasma cell dyscrasia. Abnormalities detected by FISH analysis using an IGH@/CCND1 probe set were seen in 54% with RS,and 44% with column-based. We found a statistically significant difference between the yield of abnormalities detected in paired positive cases (p = 0.0001). An additional 183 consecutive post-validation samples sorted by RS showed recurrent genetic abnormalities in 85/120 (71%) of successfully sorted samples with ≥ 1% plasma cells but in none of 63 samples in which FISH analysis was completed on samples that could not be sorted due to insufficient plasma cells upon cell sorting. The column-free method successfully sorted PC,when present in ≥ 1% of cells,for detection of abnormalities by FISH. Furthermore,our data suggest that FISH analysis should not be performed on samples with an inadequate yield at the cell selection step. View Publication

Histone deacetylase (HDAC) inhibitors (HDI) induce endoplasmic reticulum (ER) stress and apoptosis,while promoting autophagy,which promotes cancer cell survival when apoptosis is compromised. Here,we determined the in vitro and in vivo activity of the combination of the pan-HDI panobinostat and the autophagy inhibitor chloroquine against human estrogen/progesterone receptor and HER2 (triple)-negative breast cancer (TNBC) cells. Treatment of MB-231 and SUM159PT cells with panobinostat disrupted the hsp90/histone deacetylase 6/HSF1/p97 complex,resulting in the upregulation of hsp. This was accompanied by the induction of enhanced autophagic flux as evidenced by increased expression of LC3B-II and the degradation of the autophagic substrate p62. Treatment with panobinostat also induced the accumulation and colocalization of p62 with LC3B-II in cytosolic foci as evidenced by immunofluorescent confocal microscopy. Inhibition of panobinostat-induced autophagic flux by chloroquine markedly induced the accumulation of polyubiquitylated proteins and p62,caused synergistic cell death of MB-231 and SUM159PT cells,and inhibited mammosphere formation in MB-231 cells,compared with treatment with each agent alone. Finally,in mouse mammary fat pad xenografts of MB-231 cells,a tumor size-dependent induction of heat shock response,ER stress and autophagy were observed. Cotreatment with panobinostat and chloroquine resulted in reduced tumor burden and increased the survival of MB-231 breast cancer xenografts. Collectively,our findings show that cotreatment with an autophagy inhibitor and pan-HDI,for example,chloroquine and panobinostat results in accumulation of toxic polyubiquitylated proteins,exerts superior inhibitory effects on TNBC cell growth,and increases the survival of TNBC xenografts. View Publication

We previously developed an antibody-avidin fusion protein (ch128.1Av) targeting the human transferrin receptor 1 (TfR1,also known as CD71),which demonstrates direct in vitro cytotoxicity against malignant hematopoietic cells. This cytotoxicity is attributed to its ability to decrease the level of TfR1 leading to lethal iron deprivation. We now report that ch128.1Av shows the ability to bind the Fcγ receptors and the complement component C1q,suggesting that it is capable of eliciting Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity and complement-mediated cytotoxicity. In addition,in 2 disseminated multiple myeloma xenograft mouse models,we show that a single dose of ch128.1Av results in significant antitumor activity,including long-term survival. It is interesting to note that the parental antibody without avidin (ch128.1) also shows remarkable in vivo anticancer activity despite its limited in vitro cytotoxicity. Finally,we demonstrate that ch128.1Av is not toxic to pluripotent hematopoietic progenitor cells using the long-term cell-initiating culture assay suggesting that these important progenitors would be preserved in different therapeutic approaches,including the in vitro purging of cancer cells for autologous transplantation and in vivo passive immunotherapy. Our results suggest that ch128.1Av and ch128.1 may be effective in the therapy of human multiple myeloma and potentially other hematopoietic malignancies. View Publication

Stem-like cells have been isolated in tumors such as breast,lung,colon,prostate and brain. A critical issue in all these tumors,especially in glioblastoma mutliforme (GBM),is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation,progression,and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue,and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days,primary neurospheres of 150-200 μm in diameter can be observed and are ready for further passaging and expansion. View Publication

The MEK1 and MEK2 inhibitor GSK1120212 is currently in phase II/III clinical development. To identify predictive biomarkers,sensitivity to GSK1120212 was profiled for 218 solid tumor cell lines and 81 hematologic malignancy cell lines. For solid tumors,RAF/RAS mutation was a strong predictor of sensitivity. Among RAF/RAS mutant lines,co-occurring PIK3CA/PTEN mutations conferred a cytostatic response instead of a cytotoxic response for colon cancer cells that have the biggest representation of the comutations. Among KRAS mutant cell lines,transcriptomics analysis showed that cell lines with an expression pattern suggestive of epithelial-to-mesenchymal transition were less sensitive to GSK1120212. In addition,a proportion of cell lines from certain tissue types not known to carry frequent RAF/RAS mutations also seemed to be sensitive to GSK1120212. Among these were breast cancer cell lines,with triple negative breast cancer cell lines being more sensitive than cell lines from other breast cancer subtypes. We identified a single gene DUSP6,whose expression was associated with sensitivity to GSK1120212 and lack of expression associated with resistance irrelevant of RAF/RAS status. Among hematologic cell lines,acute myeloid leukemia and chronic myeloid leukemia cell lines were particularly sensitive. Overall,this comprehensive predictive biomarker analysis identified additional efficacy biomarkers for GSK1120212 in RAF/RAS mutant solid tumors and expanded the indication for GSK1120212 to patients who could benefit from this therapy despite the RAF/RAS wild-type status of their tumors. View Publication

This study evaluated the effects of a mammalian target of mTOR inhibitor everolimus alone or in combination with trastuzumab on stem cells from HER2-overexpressing primary breast cancer cells and the BT474 breast cancer cell line in vitro and in vivo. For the in vitro studies,we sorted ESA(+)CD44(+)CD24(-/low) cells as stem cells from primary breast cancer cells and BT474 cells using flow cytometry. The MTT assay was used to quantify the inhibitory effect of the drugs on total cells and stem cells specifically. Stem cell apoptosis,cell cycle distributions,and their tumorigenicity after treatment were investigated by flow cytometry or soft agar colony formation assays. For the in vivo studies,BALB/c mice were injected with BT474 stem cells,and the different treatments were administered. After necropsy,the expression of Ki67,CD31,AKT1,and phospho-AKT (Thr308) was analyzed by immunohistochemistry. For the in vitro studies,Treatment with everolimus resulted in stem cell growth inhibition in a dose-dependent manner. The combination of everolimus with trastuzumab was more effective at inhibiting cell growth (P textless 0.001) and tumorigenicity (P textless 0.001) compared with single-agent therapy. In addition,an increase in G1 cell cycle arrest and an increased population of cells in early apoptosis were seen in the combination treatment group compared with either of the single-agent groups (P textless 0.01). For the in vivo studies,everolimus plus trastuzumab therapy was much more effective at reducing tumor volume in mice compared with either single agent alone (P textless 0.05). Compared with everolimus alone,the combination of everolimus and trastuzumab reduced the expression of Ki67,AKT1,and phospho-AKT (Thr308) (P textless 0.05). We conclude that everolimus has effective inhibitory effects on HER2-overexpressing stem cells in vitro and vivo. Everolimus plus trastuzumab is a rational combination treatment that may be promising in human clinical trials. View Publication

Oral squamous cell carcinomas (OSCC) often arise from dysplastic lesions. The role of cancer stem cells in tumour initiation is widely accepted,yet the potential existence of pre-cancerous stem cells in dysplastic tissue has received little attention. Cell lines from oral diseases ranging in severity from dysplasia to malignancy provide opportunity to investigate the involvement of stem cells in malignant progression from dysplasia. Stem cells are functionally defined by their ability to generate hierarchical tissue structures in consortium with spatial regulation. Organotypic cultures readily display tissue hierarchy in vitro; hence,in this study,we compared hierarchical expression of stem cell-associated markers in dermis-based organotypic cultures of oral epithelial cells from normal tissue (OKF6-TERT2),mild dysplasia (DOK),severe dysplasia (POE-9n) and OSCC (PE/CA P J15). Expression of CD44,p75(NTR),CD24 and ALDH was studied in monolayers by flow cytometry and in organotypic cultures by immunohistochemistry. Spatial regulation of CD44 and p75(NTR) was evident for organotypic cultures of normal (OKF6-TERT2) and dysplasia (DOK and POE-9n) but was lacking for OSCC (PE/CA PJ15)-derived cells. Spatial regulation of CD24 was not evident. All monolayer cultures exhibited CD44,p75(NTR),CD24 antigens and ALDH activity (ALDEFLUOR(®) assay),with a trend towards loss of population heterogeneity that mirrored disease severity. In monolayer,increased FOXA1 and decreased FOXA2 expression correlated with disease severity,but OCT3/4,Sox2 and NANOG did not. We conclude that dermis-based organotypic cultures give opportunity to investigate the mechanisms that underlie loss of spatial regulation of stem cell markers seen with OSCC-derived cells. View Publication

The aim of the present study was to determine the effect of AZD8055 on proliferation,apoptosis and glycolysis in the human cervical cancer cell line HeLa and to investigate the underlying mechanism(s) of action. HeLa human cervical cancer cells were treated with 10 nM AZD8055 for 24,48 or 72 h. MTT was used to determine cell proliferation. Annexin V/propidium iodide staining was used to determine cell apoptosis analyzed by fluorescence-activated cell sorting (FACS). Glycolytic activity was determined by measuring the activity of the key enzyme lactate dehydrogenase (LDH) and lactate production. RNA and protein expression were examined by qRT-PCR and western blotting,respectively. Treatment with AZD8055 inhibited proliferation and glycolysis,and induced apoptosis in HeLa cells in a time-dependent manner. During the prolonged treatment with AZD8055,the phosphorylation of mammalian target of rapamycin (mTOR) C1 substrates p70S6K and phosphorylation of the mTORC2 substrate Akt were deregulated,suggesting that the activity of mTOR was downregulated. Furthermore,our study showed that the expression of miR-143 was upregulated in a time-dependent manner in HeLa cells treated with AZD8055. In summary,the present study reveals a novel antitumor mechanism of AZD8055 in HeLa human cervical cancer cells. View Publication

Transforming growth factor beta (TGF-$$) signaling has been implicated in driving tumor progression and metastasis by inducing stem cell-like features in some human cancer cell lines. In this study,we have utilized a novel murine cell line NMuMG-ST,which acquired cancer stem cell (CSC) phenotypes during spontaneous transformation of the untransformed murine mammary cell line NMuMG,to investigate the role of autocrine TGF-$$ signaling in regulating their survival,metastatic ability,and the maintenance of cancer stem cell characteristics. We have retrovirally transduced a dominant-negative TGF-$$ type II receptor (DNRII) into the NMuMG-ST cell to abrogate autocrine TGF-$$ signaling. The expression of DNRII reduced TGF-$$ sensitivity of the NMuMG-ST cells in various cell-based assays. The blockade of autocrine TGF-$$ signaling reduced the ability of the cell to grow anchorage-independently and to resist serum deprivation-induced apoptosis. These phenotypes were associated with reduced levels of active and phosphorylated AKT and ERK,and Gli1 expression suggesting that these pathways contribute to the growth and survival of this model system. More interestingly,the abrogation of autocrine TGF-$$ signaling also led to the attenuation of several features associated with mammary stem cells including epithelial-mesenchymal transition,mammosphere formation,and expression of stem cell markers. When xenografted in athymic nude mice,the DNRII cells were also found to undergo apoptosis and induced significantly lower lung metastasis burden than the control cells even though they formed similar size of xenograft tumors. Thus,our results indicate that autocrine TGF-$$ signaling is involved in the maintenance and survival of stem-like cell population resulting in the enhanced metastatic ability of the murine breast cancer cells. View Publication