Krüppel-like factor 10 modulates stem cell phenotypes of pancreatic adenocarcinoma by transcriptionally regulating notch receptors.

BACKGROUND: Pancreatic adenocarcinoma (PDAC) is well known for its rapid distant metastasis and local destructive behavior. Loss of Krüppel-like factor 10 (KLF10) contributes to distant migration of PDAC. The role of KLF10 in modulating tumorigenesis and stem cell phenotypes of PDAC is unclear. METHODS: Additional depletion of KLF10 in KC (LSL: KrasG12D; Pdx1-Cre) mice, a spontaneous murine PDAC model, was established to evaluate tumorigenesis. Tumor specimens of PDAC patients were immune-stained of KLF10 to correlate with local recurrence after curative resection. Conditional overexpressing KLF10 in MiaPaCa and stably depleting KLF10 in Panc-1 (Panc-1-pLKO-shKLF10) cells were established for evaluating sphere formation, stem cell markers expression and tumor growth. The signal pathways modulated by KLF10 for PDAC stem cell phenotypes were disclosed by microarray analysis and validated by western blot, qRT-PCR, luciferase reporter assay. Candidate targets to reverse PDAC tumor growth were demonstrated in murine model. RESULTS: KLF10, deficient in two-thirds of 105 patients with resected pancreatic PDAC, was associated with rapid local recurrence and large tumor size. Additional KLF10 depletion in KC mice accelerated progression from pancreatic intraepithelial neoplasia to PDAC. Increased sphere formation, expression of stem cell markers, and tumor growth were observed in Panc-1-pLKO-shKLF10 compared with vector control. Genetically or pharmacologically overexpression of KLF10 reversed the stem cell phenotypes induced by KLF10 depletion. Ingenuity pathway analysis and gene set enrichment analysis showed that Notch signaling molecules, including Notch receptors 3 and 4, were over-expressed in Panc-1-pLKO-shKLF10. KLF10 transcriptionally suppressed Notch-3 and -4 by competing with E74-like ETS transcription factor 3, a positive regulator, for promoter binding. Downregulation of Notch signaling, either genetically or pharmacologically, ameliorated the stem cell phenotypes of Panc-1-pLKO-shKLF10. The combination of metformin, which upregulated KLF10 expression via phosphorylating AMPK, and evodiamine, a non-toxic Notch-3 methylation stimulator, delayed tumor growth of PDAC with KLF10 deficiency in mice without prominent toxicity. CONCLUSIONS: These results demonstrated a novel signaling pathway by which KLF10 modulates stem cell phenotypes in PDAC through transcriptionally regulating Notch signaling pathway. The elevation of KLF10 and suppression of Notch signaling may jointly reduce PDAC tumorigenesis and malignant progression.

[Retrospective cohort study on period of incubation and survival among former commercial plasma donors infected with HIV in Hebei province].

OBJECTIVE: To examine the state of incubation period and survival time of former commercial plasma donors (FCPDs) infected with HIV. METHODS: All objects infected with HIV were from Hebei province and found from general investigation for FCPDs in 1995. The infector cohort by 142 cases was used to estimate incubation period. In the infector cohort, the time which infectors entered the cohort was their infection time, which was the middle value of the origin date, which was January 1, 1995. The onset of AIDS was defined as an outcome event. End point of observation was Dec 31, 2010. There were 192 months in all from beginning to end. The AIDS cohort by 57 cases was used to estimate the survival of the patients. In the patient cohort, the time of AIDS onset was defined as the time entering the cohort, and death of AIDS was defined as an outcome event. The cumulative incidence ratio, cumulative mortality, illness intensity and mortality intensity were analyzed through Kaplan-Meier. RESULTS: During the observation period, 123 cases of 142 infectors developed into AIDS, the cumulative incidence was 86.42% (123/142) and the intensity was 8.53/100 person-years and the median time of incubation period was 112.0 months (95%CI: 108.8 - 115.2). The death dates of 57 patients were from 1 to 24 months after onset. The cumulative mortality was 100%, and the intensity was 250.66/100 person-years and the median survival time was 3.0 months (95%CI: 1.8 - 4.2). It was estimated that the median time was 115.0 months (9.6 years) from infection to death. CONCLUSION: The median times of incubation and median survival time were 112.0 and 3.0 months, respectively.

Upregulating sirtuin 6 ameliorates glycolysis, EMT and distant metastasis of pancreatic adenocarcinoma with krüppel-like factor 10 deficiency.

Krüppel-like factor 10 (KLF10) is a tumor suppressor in multiple cancers. In a murine model of spontaneous pancreatic adenocarcinoma (PDAC), additional KLF10 depletion accelerated distant metastasis. However, Klf10 knockout mice, which suffer from metabolic disorders, do not develop malignancy. The mechanisms of KLF10 in PDAC progression deserve further exploration. KLF10-depleted and KLF10-overexpressing PDAC cells were established to measure epithelial-mesenchymal transition (EMT), glycolysis, and migration ability. A murine model was established to evaluate the benefit of genetic or pharmacological manipulation in KLF10-depleted PDAC cells (PDACshKLF10). Correlations of KLF10 deficiency with rapid metastasis, elevated EMT, and glycolysis were demonstrated in resected PDAC tissues, in vitro assays, and murine models. We identified sirtuin 6 (SIRT6) as an essential mediator of KLF10 that modulates EMT and glucose homeostasis. Overexpressing SIRT6 reversed the migratory and glycolytic phenotypes of PDACshKLF10 cells. Linoleic acid, a polyunsaturated essential fatty acid, upregulated SIRT6 and prolonged the survival of mice injected with PDACshKLF10. Modulating HIF1α and NFκB revealed that EMT and glycolysis in PDAC cells were coordinately regulated upstream by KLF10/SIRT6 signaling. Our study demonstrated a novel KLF10/SIRT6 pathway that modulated EMT and glycolysis coordinately via NFκB and HIF1α. Activation of KLF10/SIRT6 signaling ameliorated the distant progression of PDAC.Clinical Trial Registration: ClinicalTrials.gov. identifier: NCT01666184.

Isolation of neural stem/progenitor cells by using EGF/FGF1 and FGF1B promoter-driven green fluorescence from embryonic and adult mouse brains.

Fibroblast growth factor 1 (FGF1) and FGF2 have been shown to maintain the proliferation, self-renewal and multipotent capacities of neural stem/progenitor cells (NSPCs) in vitro. FGF1 is unique for binding to all known FGF receptors. In this study, we investigated if exogenous EGF and FGF1 could be used in the isolation of NSPCs from embryonic mouse brains. We demonstrated that EGF/FGF1-responsive cells exhibited lower proliferation rate and neurosphere formation efficiency than EGF/FGF2-responsive NSPCs. However, EGF/FGF1-responsive mouse brain cells exhibited better neural differentiation capacities than EGF/FGF2-responsive NSPCs at E11.5. Using F1BGFP reporter, we further demonstrated that F1BGFP+ cells showed similar multipotent capacities to CD133+ NSPCs, and could be induced more efficiently toward neuronal differentiation. Our results suggested that EGF/FGF1-responsive cells from E11.5 mouse brains could self-renew and have better multipotency than EGF/FGF2-responsive NSPCs. Further, CD133+ and F1BGFP+ NSPCs may also represent different subsets of NSPCs during neural development and adult neurogenesis.

Krüpple-like factor 10 regulates radio-sensitivity of pancreatic cancer via UV radiation resistance-associated gene.

BACKGROUND AND PURPOSE: Krüpple-like factor 10 (Klf10), an early response gene of TGFß, was reported to be a prognostic biomarker for pancreatic cancer survival. The role of Klf10 in predicting tumor response to cancer treatment is unknown. MATERIALS AND METHODS: Genetically manipulated MiaPaCa and Panc-1 cells were established to evaluate clonogenic survival, autophagy, apoptosis and DNA repair after radiation. The interaction between Klf10 and UV radiation resistance-associated gene (UVRAG) was demonstrated by ChiP-PCR and luciferase reporter assay. Orthotopic murine tumor model and clinical specimens were used to evaluate radio-sensitivity of pancreatic cancer. RESULTS: We found Klf10 silencing correlates with enhanced pancreatic cancer clonogenic survival and murine tumor growth after radiation. UVRAG was an essential down-stream mediator transcriptionally suppressed by Klf10. Silencing UVRAG mRNA in Klf10 depleted Panc-1 cells reversed the radio-resistant phenotypes including decreased apoptosis and enhanced DNA repair as well as autophagy. Metformin, an anti-diabetic agent, was found to increase Klf10 and suppress UVRAG expression to improve radiation cytotoxicity in pancreatic cancer. The predictive value of Klf10 in radiation response and the inverse correlation with UVRAG were confirmed in cohorts of pancreatic cancer patients. CONCLUSIONS: Klf10 is a potential biomarker in predicting and sensitizing radiation effect in pancreatic cancer.

Gene expression profiles of the aurora family kinases.

The evolutionarily conserved Aurora family kinases, a family of mitotic serine/threonine kinases, has three members in humans (Aurora-A, -B and -C). Overexpression of Aurora family members, particularly Aurora-A, has been reported in many human cancers and cell lines. In this study, we present evidence based on comparative gene expression analysis via quantitative RT-PCR to delineate the relative contributions of these kinases in 60 cell lines and statistical analysis in five different human cancer microarray datasets. The analysis demonstrated the selective upregulation of these Aurora members in various cancers. In general, Aurora-A exhibited the highest expression levels, with substantially decreased quantities of the Aurora-C transcript detected relative to Aurora-A and -B. Moreover, to characterize the roles of each Aurora member, which share many similarities, we investigated the expression profiles of the family in normal tissues and a panel of different phases of the HeLa cell cycle. Finally, both Aurora-A and -B were overexpressed in a majority of esophageal tumor tissues in comparison to the normal variants. Taken together, the results show that each Aurora member exhibits distinct expression patterns, implying that they are engaged in different biological processes to accomplish more elaborate cell physiological functions in higher organisms.

Bone marrow derived macrophages fuse with intestine stromal cells and contribute to chronic fibrosis after radiation.

BACKGROUND AND PURPOSE: Bone marrow-derived cells (BMDC) have been demonstrated to play a critical role in intestine regeneration. However, organ fibrosis was one of the major side effects of bone marrow (BM) transplantation. It warrants further investigation on the mechanisms of BM cell therapy in radiation induced intestine damage. MATERIALS AND METHODS: We established three murine models to evaluate BMDC within intestines after radiation, including cre-loxP system of transgenic mice. In vitro co-culture between murine BM with human intestine stromal cells was also performed to measure the level of fusion and fibrosis after treatment with anti-fibrotic agents or after macrophage depletion. RESULTS: Despite complete recovery of epithelial mucosa from radiation damage, we found persistent proliferation and repopulation of BMDC within the lamina propria. Fusion between BM derived monocytic and intestine stromal cells correlated with the level of fibrosis and proliferation index. Depleting macrophages genetically using CD11b-DTR mouse model or pharmacologically using clodronate liposome reduced the level of cell fusion and intestine fibrosis. CONCLUSIONS: Fibrotic cues from intestine enhance fusion between BM-derived monocytes/macrophages with intestine stromal cells. The fusion hybrids promote cell cycle re-entry, proliferation and reinforce fibrosis signal. Depleting macrophages interferes with cell fusion and ameliorates radiation-induced intestine fibrosis.

Signaling adaptor protein SH2B1 enhances neurite outgrowth and accelerates the maturation of human induced neurons.

Recent advances in somatic cell reprogramming have highlighted the plasticity of the somatic epigenome, particularly through demonstrations of direct lineage reprogramming of adult mouse and human fibroblasts to induced pluripotent stem cells (iPSCs) and induced neurons (iNs) under defined conditions. However, human cells appear to be less plastic and have a higher epigenetic hurdle for reprogramming to both iPSCs and iNs. Here, we show that SH2B adaptor protein 1ß (SH2B1) can enhance neurite outgrowth of iNs reprogrammed from human fibroblasts as early as day 14, when combined with miR124 and transcription factors BRN2 and MYT1L (IBM) under defined conditions. These SH2B1-enhanced iNs (S-IBM) showed canonical neuronal morphology, and expressed multiple neuronal markers, such as TuJ1, NeuN, and synapsin, and functional proteins for neurotransmitter release, such as GABA, vGluT2, and tyrosine hydroxylase. Importantly, SH2B1 accelerated mature process of functional neurons and exhibited action potentials as early as day 14; without SH2B1, the IBM iNs do not exhibit action potentials until day 21. Our data demonstrate that SH2B1 can enhance neurite outgrowth and accelerate the maturation of human iNs under defined conditions. This approach will facilitate the application of iNs in regenerative medicine and in vitro disease modeling.

Stem cell-based therapy in neural repair.

Cell-based therapy could aid in alleviating symptoms or even reversing the progression of neurodegenerative diseases and nerve injuries. Fibroblast growth factor 1 (FGF1) has been shown to maintain the survival of neurons and induce neurite outgrowth. Accumulating evidence suggests that combination of FGF1 and cell-based therapy is promising for future therapeutic application. Neural stem cells (NSCs), with the characteristics of self-renewal and multipotency, can be isolated from embryonic stem cells, embryonic ectoderm, and developing or adult brain tissues. For NSC clinical application, several critical problems remain to be resolved: (1) the source of NSCs should be personalized; (2) the isolation methods and protocols of human NSCs should be standardized; (3) the clinical efficacy of NSC transplants must be evaluated in more adequate animal models; and (4) the mechanism of intrinsic brain repair needs to be better characterized. In addition, the ideal imaging technique for tracking NSCs would be safe and yield high temporal and spatial resolution, good sensitivity and specificity. Here, we discuss recent progress and future development of cell-based therapy, such as NSCs, induced pluripotent stem cells, and induced neurons, in neurodegenerative diseases and peripheral nerve injuries.

[Comparative study on the effect of highly active antiretroviral therapy (HAART) in patients with HIV infection acquired by blood transfusion and paid plasma donation].

OBJECTIVE: To study the effect of HAART in patients with AIDS acquire by blood transfusion and paid plasma donation. METHODS: All AIDS patients whose disease was caused by blood transfusion and commercial plasma donation came from the domicile of Hebei Province. In the group of cases of blood transfusion in whom the infection was caused by one-time blood transfusion before and after 1995, there were 189 cases, of whom 105 cases on HAART were designated as observation group, and 84 cases who were not on HAART were designated as control group. The group of AIDS patients who were former commercial plasma donors (FCPDs) had 120 patients who were identified in the survey of 1995, of whom 63 cases on HAART were designated as observation group and 57 cases who were not on HAART were as control group. Onset dates were defined as the dates into the queue. Death due to AIDS was regarded as an outcome event. Subjects who were enrolled into the observation group and control group were epidemiologically followed up regularly. Observation was ended on December 31, 2010. RESULTS: Mortality of patients after HAART from groups of FCPDs and blood recipients were 4.42/100 person-years and 6.13/100 person-years, respectively. The survival rates of HAART groups were 80% in FCPDs for 110 months and 72% in blood recipients for 90 months, respectively. Meanwhile the mortality of no HAART groups were 182.05/100 person-years and 250.66/100 person-years, respectively. Mean survival of patients whose disease was caused by plasma donation and blood transfusion were 4 months and 3 months, respectively. CONCLUSIONS: Whether the HIV infection was caused by plasmapheresis or blood transfusion, the effects of HAART did not show difference. HAART cold reduce the death intensity and prolong survival.

[A retrospective cohort study on the natural history of AIDS caused by blood transfusion].

OBJECTIVE: To study the natural history of AIDS, caused by blood transfusion. METHODS: All HIV infections and AIDS patients were from Hebei province, including those infected through blood transfusion around 1995, that were identified as through general census of former commercial plasma donors (FCPDs). Among those objects being observed during the incubation period, 354 had HIV infections (including 142 cases infected via plasmapheresis and 212 cases caused by transfusion) but had not been treated by HAART before the onset of disease. Objects being observed during the survival period, 141 were AIDS patients (including 57 cases infected via plasmapheresis and 84 cases causes by transfusion) but had not been treated by HAART before and after the onset of disease. All infectors and AIDS patients were under follow-up on the progress of illness or death, respectively. RESULTS: By December 31, 2010, the cumulative incidence among HIV infections was 88.70% (314/354), with the incidence density as 9.14/100 person-years (314/3435.75) and the median incubation period was 113 months. Of 142 HIV infections in the blood donation group and 212 infections in the blood transfusion group, the incubation periods were 112 months and 115 months, respectively. All of the 141 patients died 34 months after the onset, with the death-strength as 204.70/100 person-years (141/68.88) and the period of survival was 4 months. Among those 57 FCPDs infections, they were all died 24 months after the onset, with the death-strength as 250.66/100 person-years (57/22.74) and the survival was 3 months. The other 84 infections who were blood recipients, all died 34 months after the onset, with the death-strength as 182.05/100 person-years (84/ 46.14) and the survival was 4 months. CONCLUSION: Through this study, we noticed that the natural history of all the AIDS patients was caused by blood transmission. It was important to evaluate the natural history of HIV epidemics among both FCPDs and blood recipients, occurred before and after 1995.

SH2B1beta enhances fibroblast growth factor 1 (FGF1)-induced neurite outgrowth through MEK-ERK1/2-STAT3-Egr1 pathway.

Genetic studies have established the crucial roles of FGF signaling, FGF-induced gene expression and morphogenesis during embryogenesis. In this study, we showed that overexpressing a signaling adaptor protein, SH2B1beta, enhanced FGF1-induced neurite outgrowth in PC12 cells. SH2B1beta has previously been shown to promote nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF)-induced neurite outgrowth, in part, through prolonging NGF and GDNF-induced signaling. To delineate how SH2B1beta promotes FGF1-induced neurite outgrowth, we examined its role in FGF1-dependent signaling. Our data suggest that SH2B1beta enhances and prolongs FGF1-induced MEK-ERK1/2 and PI3K-AKT pathways. We also provided the first evidence that FGF1 induces the phosphorylation of signal transducer and activator of transcription 3 (STAT3) at serine 727 [pSTAT3(S727)] in PC12 cells. SH2B1beta enhances this phosphorylation and the expression of the immediate early gene, Egr1. Through inhibitor assays, we have further shown that MEK-ERK1/2 is required for FGF1-induced neurite outgrowth, pSTAT3(S727) and Egr1 expression. Moreover, inhibiting Rho kinase, ROCK, enhances FGF1-induced neurite outgrowth through pSTAT3(S727)-independent manner. Taken together, our results demonstrate, for the first time, that SH2B1beta enhances FGF1-induced neurite outgrowth in PC12 cells mainly through MEK-ERK1/2-STAT3-Egr1 pathway.

Brain-specific 1B promoter of FGF1 gene facilitates the isolation of neural stem/progenitor cells with self-renewal and multipotent capacities.

Fibroblast growth factor 1 (FGF1) has been shown to maintain proliferation and self-renewal capacities of neural stem/progenitor cells (NSPCs) in vitro. We have previously identified FGF1B as the major transcript of FGF1 gene expressed exclusively in brain areas that are known to be abundant for NSPCs in vivo. The 540-bp (-540 to +31) sequence upstream of the 1B transcription start site (F1B) is sufficient to drive the expression of a heterologous luciferase reporter in cultured cells. In this study, we report a direct genetic and functional approach to isolate F1B(+) NSPCs using green fluorescent protein (GFP) reporter gene under the control of human F1B promoter. The F1B-GFP reporter could facilitate the isolation of NSPCs with self-renewal and multipotent capacities from human glioblastoma tissues, developing or adult mouse brains by fluorescence-activated cell sorting. Future work elucidating the mechanisms that control FGF1B expression will help to identify new NSPC-related genes.

[Investigation on status of HIV-1 infection among blood recipients from 1994 to 1998 in certain areas of China].

OBJECTIVE: To study the infection status of HIV-1 among blood recipients from 1994 to 1998 in certain areas of Hebei province. METHODS: A general investigation was set up among all the people in 15 townships of certain areas from November 2003 to February 2005. An epidemiological investigation was conducted among people who had received blood from donors, during 1994 and 1998. Blood samples were collected. ELISA was used in preliminary screening and Western-blot (WB) was used among people who showed a positive result in the preliminary screening. RESULTS: The infection rate of HIV-1 after blood receipt was 15.54% (92/592), and the infected persons were all appeared in five medical centers of 6 townships which located at the west part of the area. HIV-1 infection happened over the years, and reaching the zenith in the year 1995. Most of the infected persons were young women. Procreation was the main cause of blood transfusion for women and trauma was for men. CONCLUSION: A typical HIV outbreak happened in certain areas after blood transfusion in Hebei.

[Following surveillance on HIV infection after blood transfusion].

OBJECTIVE: To study epidemiological features of HIV infection after blood transfusion and the situation of transmission among members of family. METHODS: The persons infected with HIV through blood transfusion and their intrafamilial transmission in some city were analyzed and testing methods of ELISA, Western-blot, RT-PCR and subtype analyzing were used. The whole surveillance data came from residents investigation around problem medical centres and HIV monitoring network around Hebei province. RESULTS: 173 people infected with HIV after blood transfusion in some city, including 89 cases found in hospital and 84 cases in CDC, accounted for 68.7% (173/252) of all of infected persons by blood transfusion in Hebei province. The rate of intrafamilial transmission, spousal transmission and mother-to-child transmission((MTCT) were 32.0% (49/153),17.0% (26/153) and 32.7% (32/98), respectively. Most of persons infected with HIV were youth among who the female were more than the male. Childbearing and women's ailments were the main cause of blood transfusion from 1990 to 1999, and traumatism surgery took second place. Infected persons by HIV blood, whose time to diagnostic were the year from 1999 to 2009, spread over Kangtai hospital and other hospital which accounted for 45.1% (78/173) and 42.2% (73/173), respectively. The genetype of all patients were B' subtype. CONCLUSION: The medical centers at the grass-roots level in some city resulted in outbreak of infected persons by HIV blood because of having no screening test antibody of HIV for liid blood donors.

[Retrospective cohort study on the rate of mother-to-child transmission among mothers infected with human immunodeficiency virus type 1 through blood transfusion].

OBJECTIVE: To study the rate of mother-to-child transmission (MTCT) on HIV-1. METHODS: All local residents from 8 townships in a region were screened for mothers who had a history of only one blood transfusion and 63 were found HIV-1 positive. A further study on these HIV-1 positive mothers and their children was conducted with the emphasis on the date of receiving blood transfusion, date and type of nationality, history regarding breastfeeding and so on. Sera specimens from 84 children born from 63 HIV-1 positive mothers were screened, using ELISA for HIV-1 antibody, and positive specimens were confirmed by Western-blot. RESULTS: The rate of MTCT was 32.1% (27/84) for children with all risk factors related to MTCT. Another 36.8% (7/19) were related to factors on intrauterine, intrapartum and breastfeeding, 35.7% (5/14) to intrapartum and breastfeeding factors, 14.3% (2/14) to intrauterine and intrapartum factors, 37.9% (11/29) to breastfeeding factor alone. By group combination analysis, the MTCT rate was 36.9% (24/65) with breastfeeding, 11.8% (2/17) with artificial feeding, and the former was significantly higher than the latter. CONCLUSION: HIV-1 MTCT rate among mothers caused by a single blood transfusion varied with different risk factors. Breastfeeding played an important role in MTCT, appeared in our study.