Dnmt3a is downregulated by Stat5a and mediates G0/G1 arrest by suppressing the miR-17-5p/Cdkn1a axis in Jak2V617F cells.

BACKGROUND: Despite of the frequently reported Dnmt3a abormality in classical myeloproliferative neoplasms (cMPNs) patients, few research explores how the Dnmt3a is regulated by Jak2V617F mutation. In this study, we have investigated how the Dnmt3a is regulated by Jak2V617F mutation and its effects on downstream signaling pathways in cMPNs. METHODS: Specimens of Jak2V617F positive cMPN patients and normal controls were collected. Murine BaF3 cell line was used to construct cell models. Dual-Glo luciferase assays and chromatin immunoprecipitation (ChIP)-qPCR were performed to detect the impact of Stat5a on transcription activity of Dnmt3a. Soft agar colony formation assay and cell counting assay were performed to detect cell proliferation. BrdU staining and flow cytometry were used to investigate cell cycle distribution. Western blotting and quantitative reverse-transcription PCR (qPCR) were performed to detect the expression levels of genes. RESULTS: Firstly, the results of western blotting and qPCR revealed that compared with the control samples, Dnmt3a is downregulated in Jak2V617F positive samples. Then we explored the mechanism behind it and found that Dnmt3a is a downstream target of Stat5a, the transcription and translation of Dnmt3a is suppressed by the binding of aberrantly activated Stat5a with Dnmt3a promoter in Jak2V617F positive samples. We further revealed the region approximately 800 bp upstream of the first exon of the Dnmt3a promoter, which includes a gamma-activated sequence (GAS) motif of Stat5a, is the specific site that Stat5a binds to. Soft agar colony formation assay, cell counting assay, and BrdU staining and flow cytometry assay found that Dnmt3a in Jak2V617F-BaF3 cells significantly affected the cell proliferation capacity and cell cycle distribution by suppressing Cdkn1a via miR-17-5p/Cdkn1a axis and mediated G0/G1 arrest. CONCLUSIONS: Transcription and translation of Dnmt3a is downregulated by the binding of Stat5a with Dnmt3a promoter in Jak2V617F cells. The GAS motif at promoter of Dnmt3a is the exact site where the Stat5a binds to. Dnmt3a conducted G0/G1 arrest through regulating miR-17-5p/Cdkn1a axis. The axis of Stat5a/Dnmt3a/miR-17-5p/Cdkn1a potentially provides a treatment target for cMPNs.

A comprehensive genome-wide analysis of long non-coding RNA and mRNA expression profiles of JAK2V617F-positive classical myeloproliferative neoplasms.

Aberrant expression of long non-coding RNAs (lncRNAs) is involved in the progression of myeloid neoplasms, but the role of lncRNAs in the JAK2V617F-positive subtype of classical myeloproliferative neoplasms (cMPNs) remains unclear. This study was conducted to clarify the expression and regulation patterns of lncRNAs in JAK2V617F-positive cMPNs, and to explore new potential carcinogenic factors of cMPNs. Bioinformatics analysis of microarray detection and wet testing verification were performed to study the expression and regulation signature of differentially expressed lncRNAs (DELs) and related genes (DEGs) in cMPNs. The expression of lncRNAs and mRNAs were observed to significantly dysregulated in JAK2V617F-positive cMPN patients compared with the normal controls. Co-expression analysis indicated that there were significant differences of the co-expression pattern of lncRNAs and mRNAs in JAK2V617F-positive cMPN patients compared to normal controls. GO and KEGG pathway analysis of DEGs and DELs showed the involvement of several pathways previously reported to regulate the pathogenesis of leukemia and cMPNs. Cis- and trans-regulation analysis of lncRNAs showed that ZNF141, DHX29, NOC2L, MAS1L, AFAP1L1, and CPN2 were significantly cis-regulated by lncRNA ENST00000356347, ENST00000456816, hsa-mir-449c, NR_026874, TCONS_00012136, uc003lqp.2, and ENST00000456816, respectively, and DELs were mostly correlated with transcription factors including CTBP2, SUZ12, REST, STAT2, and GATA4 to jointly regulate multiple target genes. In summary, expression profiles of lncRNAs and mRNAs were significantly altered in JAK2V617F-positive cMPNs, the relative signaling pathway, co-expression, cis- and trans-regulation were regulated by dysregulation of lncRNAs and several important genes, such as ITGB3, which may act as a promising carcinogenic factor, warrant further investigation.

NOTCH-mediated ex vivo expansion of human hematopoietic stem and progenitor cells by culture under hypoxia.

Activation of NOTCH signaling in human hematopoietic stem/progenitor cells (HSPCs) by treatment with an engineered Delta-like ligand (DELTA1ext-IgG [DXI]) has enabled ex vivo expansion of short-term HSPCs, but the effect on long-term repopulating hematopoietic stem cells (LTR-HSCs) remains uncertain. Here, we demonstrate that ex vivo culture of human adult HSPCs with DXI under low oxygen tension limits ER stress in LTR-HSCs and lineage-committed progenitors compared with normoxic cultures. A distinct HSC gene signature was upregulated in cells cultured with DXI in hypoxia and, after 21 days of culture, the frequency of LTR-HSCs increased 4.9-fold relative to uncultured cells and 4.2-fold compared with the normoxia + DXI group. NOTCH and hypoxia pathways intersected to maintain undifferentiated phenotypes in cultured HSPCs. Our work underscores the importance of mitigating ER stress perturbations to preserve functional LTR-HSCs in extended cultures and offers a clinically feasible platform for the expansion of human HSPCs.

[Detection of Genetic Mutations in Primary Hypereosinophilia Patients].

OBJECTIVE: To explore the potential pathogenetic mutations of primary hypereosinophilia（HEN）by sequencing FGFR1 FLT3, MPL and JAK2 genes, and to clarify their effect on clinical manifestation and prognosis of HEN patients. METHODS: The direct DNA sequencing was employed to detect the gene mutations of FGFR1, FLT3, MPL and JAK2 in HEN patients. RESULTS: One deletion mutation (2654_2753del) within tyrosine kinase domain of FLT3 gene was found in a patient suffered from severe symptoms and ended with dismal outcome, which induced a premature stop codon (G885fsX888). For FGFR1, a new variation described as 1014_1019del AACAGT for nucleotide change was found in 19 cases, resulting in T339_V340del at the protein level. CONCLUSION: The deletion of 6 bases in the FGFR1 gene (1014_1019del AACAGT) is first reported as non-synonymous SNP (nsSNP) site in the patients with primary hypereosinophilia. Deletion mutations in the FLT3 gene may be related with malignant clinical features and poor prognosis.

Sulforaphane ameliorates glucose intolerance in obese mice via the upregulation of the insulin signaling pathway.

Sulforaphane (SFN) is a dietary component with multiple bioactivities; however, its role in obesity-related metabolic derangement remains unclear. Here, the effect of SFN on the glucose intolerance of obese mice and the underlying mechanism were determined. C57B/6J male mice were randomly divided into two groups, having free access to water and a normal-fat diet (ND, n = 6) or a high-fat diet (HFD, n = 33) for 8 weeks; thereafter twelve mice having the greatest weight gain among the HFD-fed mice were considered as obese mice. These obese mice were randomly divided into two groups and treated orally for 6 weeks with or without SFN (100 µmol per kg bw, 3 times per week). During this period the animals were continuously maintained on a ND or a HFD. Blood glucose and serum insulin were examined; then glucose tolerance and insulin resistance were evaluated. In addition, the expression of insulin signaling pathway-related genes in the muscle was determined. Our data showed that the obese mice presented a marked insulin resistance and glucose intolerance as compared to the control group, while SFN treatment exerted a prominently protective effect. In addition, the SFN-treated obese mice had a significantly increased insulin receptor substrate 1 (IRS-1) protein level (P < 0.05), markedly elevated Akt activation, as well as dramatically enhanced phosphorylation of PDK-1 (P < 0.05) when compared with the SFN-untreated obese mice. Moreover, the SFN-treated obese mice exhibited a significantly enhanced translocation of GLUT4 (P < 0.05) to the plasma membrane in the muscle compared to the obese mice without SFN treatment. In conclusion, our results support the notion that SFN acts as a promising agent to improve glucose tolerance through the up-regulation of insulin signaling mainly involving the IRS-1/Akt/GLUT4 pathway in the muscle.

Different treatment strategies and molecular features between right-sided and left-sided colon cancers.

The colon is derived from the embryological midgut and hindgut separately, with the right colon and left colon having different features with regards to both anatomical and physiological characteristics. Cancers located in the right and left colon are referred to as right colon cancer (RCC) and left colon cancer (LCC), respectively, based on their apparent anatomical positions. Increasing evidence supports the notion that not only are there differences in treatment strategies when dealing with RCC and LCC, but molecular features also vary between them, not to mention the distinguishing clinical manifestations. Disease-free survival after radical surgery of both RCC and LCC are similar. In the treatment of RCC, the benefit gained from adjuvant FOLFIRI chemotherapy is superior, or at least similar, to LCC, but inferior to LCC if FOLFOX regimen is applied. On the other hand, metastatic LCC exhibits longer survival than that of RCC in a palliative chemotherapy setting. For KRAS wild-type cancers, LCC benefits more from cetuximab treatment than RCC. Moreover, advanced LCC shows a higher sensitivity to bevacizumab treatment in comparison with advanced RCC. Significant varieties exist at the molecular level between RCC and LCC, which may serve as the cause of all apparent differences. With respect to carcinogenesis mechanisms, RCC is associated with known gene types, such as MMR, KRAS, BRAF, and miRNA-31, while LCC is associated with CIN, p53, NRAS, miRNA-146a, miRNA-147b, and miRNA-1288. Regarding protein expression, RCC is related to GNAS, NQO1, telomerase activity, P-PDH, and annexin A10, while LCC is related to Topo I, TS, and EGFR. In addition, separated pathways dominate progression to relapse in RCC and LCC. Therefore, RCC and LCC should be regarded as two heterogeneous entities, with this heterogeneity being used to stratify patients in order for them to have the optimal, current, and novel therapeutic strategies in clinical practice. Additional research is needed to uncover further differences between RCC and LCC.

Feasibility and accuracy of sentinel lymph node biopsy in clinically node-positive breast cancer after neoadjuvant chemotherapy: a meta-analysis.

Sentinel lymph node biopsy (SLNB) has replaced conventional axillary lymph node dissection (ALND) in axillary node-negative breast cancer patients. However, the use of SLNB remains controversial in patients after neoadjuvant chemotherapy (NAC). The aim of this review is to evaluate the feasibility and accuracy of SLNB after NAC in clinically node-positive patients. Systematic searches were performed in the PubMed, Embase, and Cochrane Library databases from 1993 to December 2013 for studies on node-positive breast cancer patients who underwent SLNB after NAC followed by ALND. Of 436 identified studies, 15 were included in this review, with a total of 2,471 patients. The pooled identification rate (IR) of SLNB was 89% [95% confidence interval (CI) 85-93%], and the false negative rate (FNR) of SLNB was 14% (95% CI 10-17%). The heterogeneity of FNR was analyzed by meta-regression, and the results revealed that immunohistochemistry (IHC) staining may represent an independent factor (P = 0.04). FNR was lower in the IHC combined with hematoxylin and eosin (H&E) staining subgroup than in the H&E staining alone subgroup, with values of 8.7% versus 16.0%, respectively (P = 0.001). Thus, SLNB was feasible after NAC in node-positive breast cancer patients. In addition, the IR of SLNB was respectable, although the FNR of SLNB was poor and requires further improvement. These findings indicate that IHC may improve the accuracy of SLNB.

Surgical resection of advanced gastric cancer following trastuzumab/oxaliplatin/capecitabine combination therapy.

Late-stage gastric adenocarcinoma patients have a poor prognosis because of high recurrence rates. To improve long-term outcomes, perioperative chemotherapies are combined with surgery. Human epidermal growth factor receptor 2 (HER2) overexpression had been noted in gastric cancer; therefore, trastuzumab has been used occasionally in this setting. A 63-year-old male Chinese patient, who was diagnosed with adenocarcinoma in the gastric antrum, as well as lymph node metastases along the left gastric and hepatic artery, and left adrenal area, was admitted to our hospital. HER2 expression was positive, and cluster amplification was detected in a fluorescence in situ hybridization assay. The patient received three cycles of a neoadjuvant trastuzumab/oxaliplatin /capecitabine regimen. He subsequently underwent distal gastrectomy, D2+ lymphadenectomy, left adrenalectomy, cholecystectomy and Billroth II anastomosis. Treatment was continued with another five postoperative cycles of the same medication and trastuzumab application for 1 year. No recurrence has been observed 18 mo after the operation. Trastuzumab as perioperative and adjuvant medication, in combination with oxaliplatin and capecitabine for a HER2-overexpressing advanced gastric adenocarcinoma, led to recurrence-free survival of at least 18 mo after surgery.

Rat injury model of docetaxel extravasation.

Docetaxel is a novel type of chemotherapy drug that actively treats a number of malignant tumors. The aim of the present study was to explore the severity and natural course of tissue damage induced by docetaxel extravasation and to confirm the vesicant potential of docetaxel. Rats were selected for the establishment of the ulcer model. Different volumes and concentrations were explored to induce the skin ulcer and to confirm the optimum rational injection model. The natural course of tissue injury and pathological changes produced by docetaxel extravasation were observed by comparing to vinorelbine extravasation. A 0.4 ml volume and a 6 mg/ml concentration were the optimum rational injection model for the induction of the skin ulcer. The docetaxel extravasation induced local tissue necrosis, followed by granuloma formation and hyperpigmentation or scar formation. The severity of the injury depended on the concentration of the extravasation used in the rat model. The injury occurred on the first day following extravasation and lasted 4-6 weeks. The damage from docetaxel was weaker than vinorelbine in association with the depth and extension of necrosis. In conclusion, docetaxel extravasation can induce tissue necrosis. However, the severity of necrosis was weaker than that of vinorelbine. Docetaxel has superficial vesicant properties.

Clinical characteristics and prognosis of young patients with colorectal cancer in Eastern China.

AIM: To explore the clinical characteristics and prognosis of young patients with colorectal cancer patients in Eastern China. METHODS: A total of 1335 patients with colorectal cancer treated from December 1985 to December 2005 at the Second Affiliated Hospital of Zhejiang University School of Medicine were studied retrospectively. The patients were divided into two groups, a younger group (aged ≤ 30 years) and an older group (aged > 30 years), and comparison was made in the clinical characteristics and prognosis between the two groups. Chi-square test was used for data analysis of all categorical variables, and overall survival (OS) was calculated by the Kaplan-Meier method. A multivariate analysis was performed using the Cox model. RESULTS: There were 42 (3.1%) and 1293 (96.9%) cases in the younger group and older group, respectively. Univariate analysis showed that the 5- and 10-year OS in the younger group were 33.9% and 26.1%, respectively, and those in the older group were 60.1% and 52.2%, respectively. Younger group had poor survival (χ(2) = 14.146, P = 0.000). Multivariate analysis revealed that age was not a dependent factor for prognosis (OR = 0.866, 95%CI: 0.592-1.269, P = 0.461). Stratified analysis indicated that in stage III and IV disease, the 5- and 10-year OS were 24.6% and 14.8% in the younger group, and 40.4% and 33.3% in the older group, respectively, with a significant difference between the two groups (χ(2) = 5.101, P = 0.024). In the subgroup of radical surgery, the 5- and 10-year OS were 44.3% and 34.2% in the younger group, and 69.6% and 60.5% in the older group, with a difference being significant between the two groups (χ(2) = 7.830, P = 0.005). CONCLUSION: Compared with older patients, the younger patients have lower survival, especially in the subgroups of stage III and IV disease and radical surgery.

N-Propionyl polysialic acid precursor enhances the susceptibility of multiple myeloma to antitumor effect of anti-NprPSA monoclonal antibody.

AIM: To study the antitumor effect of anti-NprPSA monoclonal antibody (mAb) in combination with ManNPr, a precursor of N-propionyl PSA, in multiple myeloma (MM), and to explore the mechanisms of the action. METHODS: Human multiple myeloma cell line RPMI-8226 was tested. The cells were pre-treated with ManNPr (1, 2, and 4 mg/mL), and then incubated with anti-NprPSA mAb (1 mg/mL). Cell apoptosis in vitro was detected using MTT assay and flow cytometry. BALB/c nude mice were inoculated sc with RPC5.4 cells. On 5 d after the injection, the mice were administered sc with anti-NprPSA mAb (200 µg/d) and ManNPr (5 mg/d) for 8 d. The tumor size and body weight were monitored twice per week. TUNEL assay was used for detecting apoptosis in vivo. The apoptotic pathway involved was examined using Western blot analysis and caspase inhibitor. RESULTS: Treatment of RPMI-8226 cells with anti-NprPSA mAb alone failed to inhibit cell growth in vitro. In RPMI-8226 cells pretreated with ManNPr, however, the mAb significantly inhibited the cell proliferation, decreased the viability, and induced apoptosis, which was associated with cleavage of caspase-3, caspase-8, caspase-9, and poly(ADP-ribose) polymerase. In the mouse xenograft model, treatment with the mAb in combination with ManNPr significantly inhibited the tumor growth, and induced significant apoptosis as compared to treatment with the mAb alone. Moreover, apoptosis induced by the mAb in vivo resulted from the activation of the caspases and poly(ADP-ribose) polymerase. CONCLUSION: The anti-NprPSA mAb in combination with ManNPr is an effective treatment for in vitro and in vivo induction of apoptosis in multiple myeloma.

[Study of hypermethylation of SOCS gene in typical myeloproliferative disease].

OBJECTIVE: To investigate the clinical role of hypermethylation of suppressor of cytokine signaling (SOCS) on typical myeloproliferative disease (MPD) patients and its mechanism. METHODS: Methylation specific PCR was used to detect SOCS1, 2, 3 methylation, direct DNA sequencing was performed to detect JAK2V617F mutation, real-time fluorescence quantitative PCR were applied to evaluate transcriptional activity of SOCS1, 2, 3. RESULTS: Among 100 MPD patients, hypermethylation of SOCS1 was detected in 27 (27%), hypermethylation of SOCS2 in 9 (9%), hypermethylation of SOCS3 in 34 (34%); JAK2V617F mutation in 64 (64%). Hypermethylation of SOCS1, 3 greatly inhibited gene expression compared with unmethylated ones (P < 0.05). Presence of JAK2V617F mutation markedly down-regulated SOCS1, 3 gene mRNA expression compared with wild JAK2V617F (P < 0.05). CONCLUSION: Hypermethylation of SOCS1, 3 and JAK2V617F mutation exist in MPD, which inhibited SOCS1, 3 gene expression. SOCS hypermethylation and JAK2V617F mutation can activate JAK-STAT signaling pathways, these observations may provide a potential therapeutic direction.

[Research advances on relationship between suppressor of cytokine signaling 3 and myeloproliferative neoplasms].

Suppressor of cytokine signaling (SOCS) is a protein family negatively regulating signal transduction pathway of a certain class of cytokines and growth factors. More than 20 members have been found in SOCS family, SOCS3 is one of them and has been studied hottest and most clearly. Recent studies demonstrated that SOCS3 abnormalities were found in patients with myeloproliferative neoplasms (MPN), suggesting that SOCS3 plays a significant role in the pathogenesis, development and metastasis in MPN. In this review, the advances of research on relationship between SOCS3 and MPN were summarized, including general profile of SOCS family; structure, function and regulation of SOCS3, relation of SOCS3 to MPN and so on.

[Efficacy of neoadjuvant chemotherapy with FEC and TEC regimen on breast cancer].

BACKGROUND AND OBJECTIVE: Neoadjuvant chemotherapy can remarkably decrease tumor size and stage, and improve the efficacy of comprehensive treatment on breast carcinoma. This study was to investigate the efficacy of neoadjuvant chemotherapy with FEC and TEC regimen on breast cancer. METHODS: From April 1998 to April 2004, 112 breast cancer patients were treated with neoadjuvant chemotherapy. Among them, 77 were treated with FEC regimen and 35 with TEC regimen for 2-3 cycles before operation. Simultaneously, 105 breast cancer patients who only received postoperative chemotherapy were regarded as control. RESULTS: All patients were followed up for 10-107 months (median, 61.4 months). After neoadjuvant chemotherapy, 31 patients (27.7%) had clinical complete remission, and the total response rate was 79.5%. The 5-year disease-free and overall survival rates were significantly higher in neoadjuvant chemotherapy group than in control group (67.2% vs. 49.4%, chi(2)=4.092, P=0.043; 84.7% vs. 69.1%, chi(2)=4.034, P=0.045). CONCLUSION: Neoadjuvant chemotherapy with FEC and TEC regimens is an effective treatment for operable breast cancers, and can prolong the long-term survival and disease-free survival after operation.

FIP1L1-PDGFRalpha alone or with other genetic abnormalities reveals disease progression in chronic eosinophilic leukemia but good response to imatinib.

BACKGROUND: The FIP1L1-PDGFRalpha fusion gene plays an important role in the pathogenesis of chronic eosinophilic leukemia (CEL) and is a direct therapeutic target of the tyrosine kinase inhibitor imatinib mesylate. METHODS: In 24 hypereosinophilic syndromes (HES) patients, using reverse transcriptase-polymerase chain reaction (RT-PCR), nested PCR and sequence analysis, we investigated the frequency of FIP1L1-PDGFRalpha and other abnormalities of tyrosine kinase family genes like PDGFRalpha, PDGFRbeta, C-KIT, FGFR1, ABL and FLT3 as well as gene mutation "hotspots", like MPL515 and JAK2V617F, frequently involved in myeloproliferative diseases. Fluorescence in situ hybridization was used to confirm the 4q12 deletion. RESULTS: The FIP1L1-PDGFRalpha fusion transcript was found in 8 (33%) of 24 patients with HES, corresponding to the chromosome 4q12 deletion identified by FISH. The FIP1L1-PDGFRalpha-associated patients diagnosed with CEL, frequently had hepatosplenomegaly, eosinophil-related tissue damage, anemia, thrombocytopenia, myelofibrosis and a short overall survival time. Nevertheless, imatinib mesylate induced rapid and complete hematological responses in treated FIP1L1-PDGFRalpha cases, followed by molecular remission and reversal of myelofibrosis. FIP1L1-PDGFRalpha fusion could co-exist with other mutations of tyrosine kinase family genes, like FLT3 or PDGFRbeta. We also demonstrated that the SNPs of PDGFRbeta were associated with selective splicing of exon 19 in case 20. CONCLUSIONS: Correlating the CEL genotype with phenotype, FIP1L1-PDGFRalpha emerges as a relatively homogeneous clinicobiological entity that co-exists with other abnormalities of tyrosine kinase family genes. It reflects the disease progression and there is a good response to imatinib. Detection of the FIP1L1-PDGFRalpha fusion gene is valid for both CEL diagnosis and therapy surveillance.

NOTCH1 mutations in T-cell acute lymphoblastic leukemia: prognostic significance and implication in multifactorial leukemogenesis.

PURPOSE: NOTCH signaling pathway is essential in T-cell development and NOTCH1 mutations are frequently present in T-cell acute lymphoblastic leukemia (T-ALL). To gain insight into its clinical significance, NOTCH1 mutation was investigated in 77 patients with T-ALL. EXPERIMENTAL DESIGN: Detection of NOTCH1 mutation was done using reverse transcription-PCR amplification and direct sequencing, and thereby compared according to the clinical/biological data of the patients. RESULTS: Thirty-two mutations were identified in 29 patients (with dual mutations in 3 cases), involving not only the heterodimerization and proline/glutamic acid/serine/threonine domains as previously reported but also the transcription activation and ankyrin repeat domains revealed for the first time. These mutations were significantly associated with elevated WBC count at diagnosis and independently linked to short survival time. Interestingly, the statistically significant difference of survival according to NOTCH1 mutations was only observed in adult patients (>18 years) but not in pediatric patients (< or = 18 years), possibly due to the relatively good overall response of childhood T-ALL to the current chemotherapy. NOTCH1 mutations could coexist with HOX11, HOX11L2, or SIL-TAL1 expression. The negative effect of NOTCH1 mutation on prognosis was potentiated by HOX11L2 but was attenuated by HOX11. CONCLUSION: NOTCH1 mutation is an important prognostic marker in T-ALL and its predictive value could be even further increased if coevaluated with other T-cell-related regulatory genes. NOTCH pathway thus acts combinatorially with oncogenic transcriptional factors on T-ALL pathogenesis.