技术资料

PTPN11 encodes the protein tyrosine phosphatase SHP-2,which relays signals from growth factor receptors to Ras and other effectors. Germline PTPN11 mutations underlie about 50% of Noonan syndrome (NS),a developmental disorder that is associated with an elevated risk of juvenile myelomonocytic leukemia (JMML). Somatic PTPN11 mutations were recently identified in about 35% of patients with JMML; these mutations introduce amino acid substitutions that are largely distinct from those found in NS. We assessed the functional consequences of leukemia-associated PTPN11 mutations in murine hematopoietic cells. Expressing an E76K SHP-2 protein induced a hypersensitive pattern of granulocyte-macrophage colony-forming unit (CFU-GM) colony growth in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3) that was dependent on SHP-2 catalytic activity. E76K SHP-2 expression also enhanced the growth of immature progenitor cells with high replating potential,perturbed erythroid growth,and impaired normal differentiation in liquid cultures. In addition,leukemia-associated SHP-2 mutations conferred a stronger phenotype than a germline mutation found in patients with NS. Mutant SHP-2 proteins induce aberrant growth in multiple hematopoietic compartments,which supports a primary role of hyperactive Ras in the pathogenesis of JMML. View Publication

Recent studies indicate that dysregulation of the Akt/PKB family of serine/threonine kinases is a prominent feature of many human cancers. The Akt/PKB family is composed of three members termed Akt1/PKBalpha,Akt2/PKBbeta,and Akt3/PKBgamma. It is currently not known to what extent there is functional overlap between these family members. We have recently identified small molecule inhibitors of Akt. These compounds have pleckstrin homology domain-dependent,isozyme-specific activity. In this report,we present data showing the relative contribution that inhibition of the different isozymes has on the apoptotic response of tumor cells to a variety of chemotherapies. In multiple cell backgrounds,maximal induction of caspase-3 activity is achieved when both Akt1 and Akt2 are inhibited. This induction is not reversed by overexpression of functionally active Akt3. The level of caspase-3 activation achieved under these conditions is equivalent to that observed with the phosphatidylinositol-3-kinase inhibitor LY294002. We also show that in different tumor cell backgrounds inhibition of mammalian target of rapamycin,a downstream substrate of Akt,is less effective in inducing caspase-3 activity than inhibition of Akt1 and Akt2. This shows that the survival phenotype conferred by Akt can be mediated by signaling pathways independent of mammalian target of rapamycin in some tumor cell backgrounds. Finally,we show that inhibition of both Akt1 and Akt2 selectively sensitizes tumor cells,but not normal cells,to apoptotic stimuli. View Publication

Overwhelming evidence from leukemia research has shown that the clonal population of neoplastic cells exhibits marked heterogeneity with respect to proliferation and differentiation. There are rare stem cells within the leukemic population that possess extensive proliferation and self-renewal capacity not found in the majority of the leukemic cells. These leukemic stem cells are necessary and sufficient to maintain the leukemia. Interestingly,the BCR/ABL fusion gene,which is present in chronic myelogenous leukemia (CML),was also detected in the endothelial cells of patients with CML,suggesting that CML might originate from hemangioblastic progenitor cells that can give rise to both blood cells and endothelial cells. Here we isolated fetal liver kinase-1-positive (Flk1+) cells carrying the BCR/ABL fusion gene from the bone marrow of 17 Philadelphia chromosome-positive (Ph+) patients with CML and found that these cells could differentiate into malignant blood cells and phenotypically defined endothelial cells at the single-cell level. These findings provide direct evidence for the first time that rearrangement of the BCR/ABL gene might happen at or even before the level of hemangioblastic progenitor cells,thus resulting in detection of the BCR/ABL fusion gene in both blood and endothelial cells. View Publication

The identification of succinate dehydrogenase (SDH),fumarate hydratase (FH) and isocitrate dehydrogenase (IDH) mutations in human cancers has rekindled the idea that altered cellular metabolism can transform cells. Inactivating SDH and FH mutations cause the accumulation of succinate and fumarate,respectively,which can inhibit 2-oxoglutarate (2-OG)-dependent enzymes,including the EGLN prolyl 4-hydroxylases that mark the hypoxia inducible factor (HIF) transcription factor for polyubiquitylation and proteasomal degradation. Inappropriate HIF activation is suspected of contributing to the pathogenesis of SDH-defective and FH-defective tumours but can suppress tumour growth in some other contexts. IDH1 and IDH2,which catalyse the interconversion of isocitrate and 2-OG,are frequently mutated in human brain tumours and leukaemias. The resulting mutants have the neomorphic ability to convert 2-OG to the (R)-enantiomer of 2-hydroxyglutarate ((R)-2HG). Here we show that (R)-2HG,but not (S)-2HG,stimulates EGLN activity,leading to diminished HIF levels,which enhances the proliferation and soft agar growth of human astrocytes. These findings define an enantiomer-specific mechanism by which the (R)-2HG that accumulates in IDH mutant brain tumours promotes transformation and provide a justification for exploring EGLN inhibition as a potential treatment strategy. View Publication

INTRODUCTION: Retinoic acid signaling plays key roles in embryonic development and in maintaining the differentiated status of adult tissues. Recently,the nuclear retinoic acid receptor (RAR) isotypes α,β and γ were found to play specific functions in the expansion and differentiation of the stem compartments of various tissues. For instance,RARγ appears to be involved in stem cell compartment expansion,while RARα and RARβ are implicated in the subsequent cell differentiation. We found that over-expressing c-Myc in normal mouse mammary epithelium and in a c-Myc-driven transgenic model of mammary cancer,disrupts the balance between RARγ and RARα/β in favor of RARγ. METHODS: The effects of c-Myc on RAR isotype expression were evaluated in normal mouse mammary epithelium,mammary tumor cells obtained from the MMTV-Myc transgenic mouse model as well as human normal immortalized breast epithelial and breast cancer cell lines. The in vivo effect of the RARα-selective agonist 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)carboxamido]benzoic acid (Am580) was examined in the MMTV-Myc mouse model of mammary tumorigenesis. RESULTS: Modulation of the RARα/β to RARγ expression in mammary glands of normal mice,oncomice,and human mammary cell lines through the alteration of RAR-target gene expression affected cell proliferation,survival and tumor growth. Treatment of MMTV-Myc mice with the RARα-selective agonist Am580 led to significant inhibition of mammary tumor growth (˜90%,Ptextless0.001),lung metastasis (Ptextless0.01) and extended tumor latency in 63% of mice. Immunocytochemical analysis showed that in these mice,RARα responsive genes such as Cyp26A1,E-cadherin,cellular retinol-binding protein 1 (CRBP1) and p27,were up-regulated. In contrast,the mammary gland tumors of mice that responded poorly to Am580 treatment (37%) expressed significantly higher levels of RARγ. In vitro experiments indicated that the rise in RARγ was functionally linked to promotion of tumor growth and inhibition of differentiation. Thus,activation of the RARα pathway is linked to tumor growth inhibition,differentiation and cell death. CONCLUSIONS: The functional consequence of the interplay between c-Myc oncogene expression and the RARγ to RARα/β balance suggests that prevalence of RARγ over-RARα/β expression levels in breast cancer accompanied by c-Myc amplification or over-expression in breast cancer should be predictive of response to treatment with RARα-isotype-specific agonists and warrant monitoring during clinical trials. View Publication

BACKGROUND Patients with gene expression profiling-defined high-risk myeloma in relapse have poor outcomes with current therapies. We tested whether natural killer cells expanded by co-culture with K562 cells transfected with 41BBL and membrane-bound interleukin-15 could kill myeloma cells with a high-risk gene expression profile in vitro and in a unique model which recapitulates human myeloma. DESIGN AND METHODS OPM2 and high-risk primary myeloma tumors were grown in human fetal bone implanted into non-obese diabetic severe combined immunodeficiency mice with a deficient interleukin-2 receptor gamma chain. These mice are devoid of endogenous natural killer and T-cell activity and were used to determine whether adoptively transferred expanded natural killer cells could inhibit myeloma growth and myeloma-associated bone destruction. RESULTS Natural killer cells from healthy donors and myeloma patients expanded a median of 804- and 351-fold,respectively,without significant T-cell expansion. Expanded natural killer cells killed both allogeneic and autologous primary myeloma cells avidly via a perforin-mediated mechanism in which the activating receptor NKG2D,natural cytotoxicity receptors,and DNAX-accessory molecule-1 played a central role. Adoptive transfer of expanded natural killer cells inhibited the growth of established OPM2 and high-risk primary myeloma tumors grown in the murine model. The transferred,expanded natural killer cells proliferated in vivo in an interleukin-2 dose-dependent fashion,persisted up to 4 weeks,were readily detectable in the human bone,inhibited myeloma growth and protected bone from myeloma-induced osteolysis. CONCLUSIONS These studies provide the rationale for testing expanded natural killer cells in humans. View Publication

BACKGROUND: Recurrence is a common problem in bladder cancer; this has been attributed to cancer stem cells. In this study,we characterized potential cancer stem cell populations isolated from three cell lines that demonstrate different responses to cisplatin. MATERIALS AND METHODS: The ALDEFLUOR® assay was used to isolate cells from TCCSUP,T24,and 5637 cell lines,and these cells were evaluated for their ability to form colonies,differentiate,migrate and invade. RESULTS: The cell lines demonstrate a spectrum of aldehyde dehydrogenase high (ALDH(High)) populations that correlate with resistance to cisplatin. In the two resistant cell lines,T24 and 5637,the ALDH(High) cells demonstrate increased colony formation,migration,invasion,and ability to differentiate. The resistant T24 and 5637 cell lines may serve as models to investigate alternative therapies for bladder cancer. View Publication

The anti-diabetic drug metformin is rapidly emerging as a potential anti-cancer agent. Metformin,effective in treating type 2 diabetes and the insulin resistance syndromes,improves insulin resistance by reducing hepatic gluconeogenesis and by enhancing glucose uptake by skeletal muscle. Epidemiological studies have consistently associated metformin use with decreased cancer incidence and cancer-related mortality. Furthermore,numerous preclinical and clinical studies have demonstrated anti-cancer effects of metformin,leading to an explosion of interest in evaluating this agent in human cancer. The effects of metformin on circulating insulin levels indicate a potential efficacy towards cancers associated with hyperinsulinaemia; however,metformin may also directly inhibit tumour growth. In this review,we describe the mechanism of action of metformin and summarise the epidemiological,clinical and preclinical evidence supporting a role for metformin in the treatment of cancer. In addition,the challenges associated with translating preclinical results into therapeutic benefit in the clinical setting will be discussed. View Publication

The aim is to investigate the clinical implications of the Oct-4 and Nestin protein in human breast cancers. A total of 346 cases including 26 fresh and 320 paraffin-embedded tumor tissues were selected for characterizing the frequency of CD44(+)CD24(-) tumor cells by flow cytometry and the differential expression of the stem cell-related genes between CD44(+)CD24(-) and non-CD44(+)CD24(-) tumor cells was analyzed by PCR Array and immunofluorescence. In comparison with the non-CD44(+)CD24(-) tumor cells,the CD44(+)CD24(-),particularly for those with high percentage of Oct-4(+) and Nestin(+),tumor cells had higher tumorigenicity by forming mammospheres in vitro. More importantly,42 (13.125%) out of 320 tumor tissues were positive for Oct-4 and Nestin staining. Universal analysis and multivariate analysis revealed that the expression of Oct-4 and Nestin was associated significantly with younger age,pathogenic degrees,lymph node metastasis and triple-negative breast cancer independently (P textless 0.05) as well as shorter survival (P = 0.001). Oct-4 and Nestin were important regulators of the development of breast cancer,and Oct-4 and Nestin may be used as predictors for the prognosis of breast cancers. View Publication

ETV6-RUNX1 gene fusion is usually an early,prenatal event in childhood acute lymphoblastic leukemia (ALL). Transformation results in the generation of a persistent (> 14 years) preleukemic clone,which postnatally converts to ALL after the acquisition of necessary secondary genetic alterations. Many cancer cells show some expression of the erythropoietin receptor (EPOR) gene,although the functionality" of any EPOR complexes and their relevant signaling pathways in nonerythroid cells has not been validated. EPOR mRNA is selectively and ectopically expressed in ETV6-RUNX1(+) ALL but the presence of a functional EPOR on the cell surface and its role in leukemogenesis driven by ETV6-RUNX1 remains to be identified. Here we show that ETV6-RUNX1 directly binds the EPOR promoter and that expression of ETV6-RUNX1 alone in normal pre-B cells is sufficient to activate EPOR transcription. We further reveal that murine and human ETV6-RUNX1(+) cells expressing EPOR mRNA have EPO ligand binding activity that correlates with an increased cell survival through activation of the JAK2-STAT5 pathway and up-regulation of antiapoptotic BCL-XL. These data support the contention that ETV6-RUNX1 directly activates ectopic expression of a functional EPOR and provides cell survival signals that may contribute critically to persistence of covert premalignant clones in children. View Publication

Breast cancer exhibits a propensity to metastasize to bone,resulting in debilitating skeletal complications associated with significant morbidity and poor prognosis. The cross-talk between metastatic cancer cells and bone is critical to the development and progression of bone metastases. We have shown the involvement of the HGF/c-MET system in tumor-bone interaction contributing to human breast cancer metastasis. Therefore,disruption of HGF/c-MET signaling is a potential targeted approach to treating metastatic bone disease. In this study,we evaluated the effects of c-MET inhibition by both an oral,selective,small-molecule c-MET inhibitor,tivantinib,and a specific short hairpin RNA (shRNA) against c-MET in a mouse model of human breast cancer. Tivantinib exhibited dose-dependent antimetastatic activity in vivo,and the 120 mg/kg dose,proven to be suboptimal in reducing subcutaneous tumor growth,induced significant inhibition of metastatic growth of breast cancer cells in bone and a noteworthy reduction of tumor-induced osteolysis. shRNA-mediated c-MET silencing did not affect in vitro proliferation of bone metastatic cells,but significantly reduced their migration,and this effect was further enhanced by tivantinib. Both observations were confirmed in vivo. Indeed,more pronounced tumor growth suppression with concomitant marked decreases of lytic lesions and prolongation of survival were achieved by dual c-MET inhibition using both tivantinib and RNA interference strategies. Overall,our findings highlighted the effectiveness of c-MET inhibition in delaying the onset and progression of bone metastases and strongly suggest that targeting c-MET may have promising therapeutic value in the treatment of bone metastases from breast cancer. View Publication

We investigated the expression status of periostin in breast cancer stem cells and its clinical implications in order to lay a foundation for managing breast cancer. CD44+/CD24-/line- tumor cells (CSC) from clinical specimens were sorted using flow cytometry. Periostin expression status was detected in CSC cells and 1,086 breast cancer specimens by Western blot and immunohistochemistry staining,with the CSC ratio determined by immunofluorescence double staining. The relationship between the periostin protein and clinico-pathological parameters and prognosis was subsequently determined. As a result,CSC cells are more likely to generate new tumors in mice and cell microspheres that are deficient in NOD/SCID compared to the control group. Periostin protein was expressed higher in CSC cells compared to the control cells and was found to be related to CSC chemotherapy resistance. Moreover,periostin expression was found to be related to the CSC ratio in 1,086 breast cancer specimens (P = 0.001). In total,334 (30.76%) of the 1,086 breast cases showed high periostin expression. After universal and Spearman regression correlation analysis,periostin was observed to be related to histological grade,CSC ratio,lymph node metastasis,tumor size,and triple-negative breast cancer (all Ptextless0.05). Furthermore,periostin was shown to attain a significantly more distant bone metastasis and worse disease-specific survival than those with none or low-expressed periostin protein (P = 0.001). In the Cox regression test,periostin protein was detected as an independent prognostic factor (P = 0.001). In conclusion,periostin was found to be related to the CSC and an independent prognostic factor for breast cancer. It is also perhaps a potential target to breast cancer. View Publication