Oncogenic Kras Initiates Leukemia in Hematopoietic Stem Cells

How oncogenes modulate the self-renewal properties of cancer-initiating cells is incompletely understood. Activating KRAS and NRAS mutations are among the most common oncogenic lesions detected in human cancer, and occur in myeloproliferative disorders (MPDs) and leukemias. We investigated the effects of expressing oncogenic Kras^G12D from its endogenous locus on the proliferation and tumor-initiating properties of murine hematopoietic stem and progenitor cells. MPD could be initiated by Kras^G12D expression in a highly restricted population enriched for hematopoietic stem cells (HSCs), but not in common myeloid progenitors. Kras^G12D HSCs demonstrated a marked in vivo competitive advantage over wild-type cells. Kras^G12D expression also increased the fraction of proliferating HSCs and reduced the overall size of this compartment. Transplanted Kras^G12D HSCs efficiently initiated acute T-lineage leukemia/lymphoma, which was associated with secondary Notch1 mutations in thymocytes. We conclude that MPD-initiating activity is restricted to the HSC compartment in Kras^G12D mice, and that distinct self-renewing populations with cooperating mutations emerge during cancer progression.     

PTK6 Promotes Cancer Migration and Invasion in Pancreatic Cancer Cells Dependent on ERK Signaling

Protein Tyrosine Kinase 6 (PTK6) is a non-receptor type tyrosine kinase that may be involved in some cancers. However, the biological role and expression status of PTK6 in pancreatic cancer is unknown. Therefore in this study, we evaluated the functional role of PTK6 on pancreatic cancer invasion. Five pancreatic cancer cell lines expressed PTK6 at varying levels. PTK6 expression was also observed in human pancreatic adenocarcinomas. PTK6 suppression by siRNA significantly reduced both cellular migration and invasion (0.59/0.49 fold for BxPC3, 0.61/0.62 for Panc1, 0.42/0.39 for MIAPaCa2, respectively, p<0.05 for each). In contrast, forced overexpression of PTK6 by transfection of a PTK6 expression vector in Panc1 and MIAPaCa2 cells increased cellular migration and invasion (1.57/1.67 fold for Panc1, 1.44/1.57 for MIAPaCa2, respectively, p<0.05). Silencing PTK6 reduced ERK1/2 activation, but not AKT or STAT3 activation, while PTK6 overexpression increased ERK1/2 activation. U0126, a specific inhibitor of ERK1/2, completely abolished the effect of PTK6 overexpression on cellular migration and invasion. These results suggest that PTK6 regulates cellular migration and invasion in pancreatic cancer via ERK signaling. PTK6 may be a novel therapeutic target for pancreatic cancer.     

卵巢移植技术应用于胰腺癌转基因小鼠模型繁殖传代研究

目的研究卵巢移植在胰腺癌转基因小鼠模型保种传代中的应用。方法通过显微手术将SV40T阳性转基因小鼠的卵巢原位移植给相同背景品系去除卵巢的正常FVB雌鼠体内。移植后与正常FVB雄鼠配种,后代以PCR检测确定阳性,统计后代发病情况。并对移植受体鼠进行卵巢的组织切片观察。结果实验中移植用供体鼠19只。成功移植给32只受体。其中有21只移植受体怀孕产子,得到后代159只,存活96只,PCR检测阳性34只,阳性率约为25.4%。结论卵巢移植可被用于转基因小鼠的传代繁殖保种。     

Tamoxifen-Induced Cre-loxP Recombination Is Prolonged in Pancreatic Islets of Adult Mice

Tamoxifen (Tm)-inducible Cre recombinases are widely used to perform gene inactivation and lineage tracing studies in mice. Although the efficiency of inducible Cre-loxP recombination can be easily evaluated with reporter strains, the precise length of time that Tm induces nuclear translocation of CreER(Tm) and subsequent recombination of a target allele is not well defined, and difficult to assess. To better understand the timeline of Tm activity in vivo, we developed a bioassay in which pancreatic islets with a Tm-inducible reporter (from Pdx1(PB)-CreER(Tm);R26R(lacZ) mice) were transplanted beneath the renal capsule of adult mice previously treated with three doses of 1 mg Tm, 8 mg Tm, or corn oil vehicle. Surprisingly, recombination in islet grafts, as assessed by expression of the β-galactosidase (β-gal) reporter, was observed days or weeks after Tm treatment, in a dose-dependent manner. Substantial recombination occurred in islet grafts long after administration of 3×8 mg Tm: in grafts transplanted 48 hours after the last Tm injection, 77.9±0.4% of β-cells were β-gal+; in β-cells placed after 1 week, 46.2±5.0% were β-gal+; after 2 weeks, 26.3±7.0% were β-gal+; and after 4 weeks, 1.9±0.9% were β-gal+. Islet grafts from mice given 3×1 mg Tm showed lower, but notable, recombination 48 hours (4.9±1.7%) and 1 week (4.5±1.9%) after Tm administration. These results show that Tm doses commonly used to induce Cre-loxP recombination may continue to label significant numbers of cells for weeks after Tm treatment, possibly confounding the interpretation of time-sensitive studies using Tm-dependent models. Therefore, investigators developing experimental approaches using Tm-inducible systems should consider both maximal recombination efficiency and the length of time that Tm-induced Cre-loxP recombination occurs.     

The Metastatic Potential and Chemoresistance of Human Pancreatic Cancer Stem Cells

Cancer stem cells (CSCs) typically have the capacity to evade chemotherapy and may be the principal source of metastases. CSCs for human pancreatic ductal carcinoma (PDAC) have been identified, but neither the metastatic potential nor the chemoresistance of these cells has been adequately evaluated. We have addressed these issues by examining side-population (SP) cells isolated from the Panc-1 and BxPC3 lines of human PDAC cells, the oncogenotypes of which differ. SP cells could be isolated from monolayers of Panc-1, but only from spheroids of BxPC3. Using orthotopic xenografts into the severely immunocompromised NSG mouse, we found that SP cells isolated from both cell lines produced tumors that were highly metastatic, in contrast to previous experience with PDAC cell lines. SP cells derived from both cell lines expressed the ABCG2 transporter, which was demonstrably responsible for the SP phenotype. SP cells gave rise to non-SP (NSP) cells in vitro and in vivo, a transition that was apparently due to posttranslational inhibition of the ABCG2 transporter. Twenty-two other lines of PDAC cells also expressed ABCG2. The sensitivity of PDAC SP cells to the vinca alkaloid vincristine could be greatly increased by verapamil, a general inhibitor of transporters. In contrast, verapamil had no effect on the killing of PDAC cells by gemcitabine, the current first-line therapeutic for PDAC. We conclude that the isolation of SP cells can be a convenient and effective tool for the study of PDAC CSCs; that CSCs may be the principal progenitors of metastasis by human PDAC; that the ABCG2 transporter is responsible for the SP phenotype in human PDAC cells, and may be a ubiquitous source of drug-resistance in PDAC, but does not confer resistance to gemcitabine; and that inhibition of ABCG2 might offer a useful adjunct in a therapeutic attack on the CSCs of PDAC.     

Role of Kras Status in Patients with Metastatic Colorectal Cancer Receiving First-Line Chemotherapy plus Bevacizumab: A TTD Group Cooperative Study

Background

In the MACRO study, patients with metastatic colorectal cancer (mCRC) were randomised to first-line treatment with 6 cycles of capecitabine and oxaliplatin (XELOX) plus bevacizumab followed by either single-agent bevacizumab or XELOX plus bevacizumab until disease progression. An additional retrospective analysis was performed to define the prognostic value of tumour KRAS status on progression-free survival (PFS), overall survival (OS) and response rates.

Methodology/Principal Findings

KRAS data (tumour KRAS status and type of mutation) were collected by questionnaire from participating centres that performed KRAS analyses. These data were then cross-referenced with efficacy data for relevant patients in the MACRO study database. KRAS status was analysed in 394 of the 480 patients (82.1%) in the MACRO study. Wild-type (WT) KRAS tumours were found in 219 patients (56%) and mutant (MT) KRAS in 175 patients (44%). Median PFS was 10.9 months for patients with WT KRAS and 9.4 months for patients with MT KRAS tumours (p = 0.0038; HR: 1.40; 95% CI:1.12–1.77). The difference in OS was also significant: 26.7 months versus 18.0 months for WT versus MT KRAS, respectively (p = 0.0002; HR: 1.55; 95% CI: 1.23–1.96). Univariate and multivariate analyses showed that KRAS was an independent variable for both PFS and OS. Responses were observed in 126 patients (57.5%) with WT KRAS tumours and 76 patients (43.4%) with MT KRAS tumours (p = 0.0054; OR: 1.77; 95% CI: 1.18–2.64).

Conclusions/Significance

This analysis of the MACRO study suggests a prognostic role for tumour KRAS status in patients with mCRC treated with XELOX plus bevacizumab. For both PFS and OS, KRAS status was an independent factor in univariate and multivariate analyses.     

Adiponectin Mediated MHC Class II Mismatched Cardiac Graft Rejection in Mice Is IL-4 Dependent

Background

Adiponectin regulates glucose and fatty-acid metabolism but its role in chronic graft rejection mediated by Th2 cytokines remains ill-defined.

Methodology/Principal Findings

Wild type and adiponectin-null mice were used as graft recipients in mouse MHC class II disparate cardiac transplantation (bm12 toB6) and the graft rejection was monitored. In adiponectin-null mice we observed that the cellular infiltrate of eosinophils, CD4⁺ and CD8⁺ T cells was reduced in grafts compared to the controls as was collagen deposition and vessel occlusion. A similar outcome was observed for skin transplants except that neutrophil infiltration was increased. Low levels of IL-4 were detected in the grafts and serum. The effect of adiponectin signaling on IL-4 expression was further investigated. Treatment with AMPK and p38 MAPK inhibitors blocked adiponectin enhanced T cell proliferation in mixed lymphocyte reactions. Inhibition of AMPK reduced eosinophil infiltration in skin grafts in wild type recipients and in contrast AMPK activation increased eosinophils in adiponectin-null recipients. The addition of adiponectin increased IL-4 production by the T cell line EL4 with augmented nuclear GATA-3 and phospho-STAT6 expression which were suppressed by knockdown of adiponectin receptor 1 and 2.

Conclusions

Our results demonstrate a direct effect of adiponectin on IL-4 expression which contributes to Th2 cytokine mediated rejection in mouse MHC class II histoincompatible transplants. These results add to our understanding of the interrelationship of metabolism and immune regulation and raise the possibility that AMPK inhibitors may be beneficial in selected types of rejection.     

EGF-Induced Acetylation of Heterogeneous Nuclear Ribonucleoproteins Is Dependent on KRAS Mutational Status in Colorectal Cancer Cells

KRAS mutational status is considered a negative predictive marker of the response to anti-EGFR therapies in colorectal cancer (CRC) patients. However, conflicting data exist regarding the variable response to EGFR-targeted therapy. The effects of oncogenic KRAS on downstream targets were studied in cell lines with different KRAS mutations. Cells harboring a single KRAS^G13D allele showed the most tumorigenic profile, with constitutive activation of the downstream pathway, rendering them EGF-unresponsive. Conversely, KRAS^A146T cells showed a full EGF-response in terms of signal transduction pathways, cell proliferation, migration or adhesion. Moreover, the global acetylome of CRC cells was also dependent on KRAS mutational status. Several hnRNP family members were identified within the 36 acetylated-proteins. Acetylation status is known to be involved in the modulation of EGF-response. In agreement with results presented herein, hnRNPA1 and L acetylation was induced in response to EGF in KRAS^A146T cells, whereas acetyl-hnRNPA1 and L levels remained unchanged after growth factor treatment in KRAS^G13D unresponsive cells. Our results showed that hnRNPs induced-acetylation is dependent on KRAS mutational status. Nevertheless hnRNPs acetylation might also be the point where different oncogenic pathways converge.     

Laminins in Metastatic Cancer

Laminins are a family of extracellular heterotrimeric glycoproteins that are the main structural component of basement membranes (BMs), perform a barrier function, and are important for adhesion, differentiation, migration, and resistance to apoptosis of various cells, including cancer cells. The review summarizes the current knowledge of how laminins produced by cancer and normal cells influence the key stages of carcinogenesis. Laminin 332 (LN-332) and LN-111 enhance proliferation of certain cancer cells and increase the tumour growth. LN-111 increases resistance to apoptosis, induces differentiation, and inhibits the epithelial–mesenchymal transition (EMT) of cancer cells. LN-332 is associated with higher adhesion and higher migration potential of cancer cells. LN-411 and LN-421 significantly increase motility of cancer cells. LN-332 and LN-511 facilitate cell–cell adhesion and affect the efficacy of cell–cell interactions. The laminin chains α4 and α5 are important for the development and function of blood and lymphatic vessels. The expression ratio of the α4 and α5 laminin chains defines the BM permeability to leukocytes and, presumably, cancer cells in blood and lymphatic vessels. Interactions between LN-511 and α2-containing laminins enhance selfrenewal and survival of circulating cancer stem cells. Moreover, laminins are involved in the formation of premetastatic niches and new colonies. Endogenous expression of the α4 laminin chain stimulates proliferation of individualised circulating cancer cells in vitro and in vivo and facilitates micrometastasis.     

EMAST Is Associated with a Poor Prognosis in Microsatellite Instable Metastatic Colorectal Cancer

Purpose

To determine the frequency and prognostic value of elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) in metastatic colorectal cancer (mCRC) patients in relation to microsatellite instability (MSI) status and MSH3 protein expression.

Material and Methods

The frequency of EMAST was evaluated in mCRC patients with MSI tumors and microsatellite stable (MSS) tumors. A literature overview was performed to compare the frequency of EMAST in our study with existing data. Immunohistochemistry for MSH3 was compared with EMAST status. Outcome was studied in terms of overall survival (OS) of mCRC patients with MSI and MSS tumors.

Results

EMAST was evaluated in 89 patients with MSI tumors (including 39 patients with Lynch syndrome) and 94 patients with MSS tumors. EMAST was observed in 45.9% (84 out of 183) of patients, with an increased frequency in MSI tumors (79.8% versus 13.8%, p < 0.001). We found no correlation between EMAST and MSH3 protein expression. There was no effect of EMAST on prognosis in patients with MSS tumors, but patients with MSI / non-EMAST tumors had a significantly better prognosis than patients with MSI / EMAST tumors (OS: HR 3.22, 95% CI 1.25-8.30).

Conclusion

Frequency of EMAST was increased in mCRC patients with MSI tumors, compared to MSS tumors. Our data suggest that the presence of EMAST correlates with worse OS in these patients. There was no effect of EMAST on the prognosis of patients with MSS tumors. A limitation of our study is the small number of patients in our subgroup analysis.     

The Ketogenic Diet and Hyperbaric Oxygen Therapy Prolong Survival in Mice with Systemic Metastatic Cancer

Introduction

Abnormal cancer metabolism creates a glycolytic-dependency which can be exploited by lowering glucose availability to the tumor. The ketogenic diet (KD) is a low carbohydrate, high fat diet which decreases blood glucose and elevates blood ketones and has been shown to slow cancer progression in animals and humans. Abnormal tumor vasculature creates hypoxic pockets which promote cancer progression and further increase the glycolytic-dependency of cancers. Hyperbaric oxygen therapy (HBO₂T) saturates tumors with oxygen, reversing the cancer promoting effects of tumor hypoxia. Since these non-toxic therapies exploit overlapping metabolic deficiencies of cancer, we tested their combined effects on cancer progression in a natural model of metastatic disease.

Methods

We used the firefly luciferase-tagged VM-M3 mouse model of metastatic cancer to compare tumor progression and survival in mice fed standard or KD ad libitum with or without HBO₂T (2.5 ATM absolute, 90 min, 3x/week). Tumor growth was monitored by in vivo bioluminescent imaging.

Results

KD alone significantly decreased blood glucose, slowed tumor growth, and increased mean survival time by 56.7% in mice with systemic metastatic cancer. While HBO₂T alone did not influence cancer progression, combining the KD with HBO₂T elicited a significant decrease in blood glucose, tumor growth rate, and 77.9% increase in mean survival time compared to controls.

Conclusions

KD and HBO₂T produce significant anti-cancer effects when combined in a natural model of systemic metastatic cancer. Our evidence suggests that these therapies should be further investigated as potential non-toxic treatments or adjuvant therapies to standard care for patients with systemic metastatic disease.     

Cell Stiffness Is a Biomarker of the Metastatic Potential of Ovarian Cancer Cells

The metastatic potential of cells is an important parameter in the design of optimal strategies for the personalized treatment of cancer. Using atomic force microscopy (AFM), we show, consistent with previous studies conducted in other types of epithelial cancer, that ovarian cancer cells are generally softer and display lower intrinsic variability in cell stiffness than non-malignant ovarian epithelial cells. A detailed examination of highly invasive ovarian cancer cells (HEY A8) relative to their less invasive parental cells (HEY), demonstrates that deformability is also an accurate biomarker of metastatic potential. Comparative gene expression analyses indicate that the reduced stiffness of highly metastatic HEY A8 cells is associated with actin cytoskeleton remodeling and microscopic examination of actin fiber structure in these cell lines is consistent with this prediction. Our results indicate that cell stiffness may be a useful biomarker to evaluate the relative metastatic potential of ovarian and perhaps other types of cancer cells.     

Circadian Phase Dependent Pharmacokinetics of Disopyramide in Mice

The focus of the reported work is investigation of disopyramide chronopharmacokinetics in the mouse. Different groups of male NMRI mice maintained under controlled environmental conditions (LD: 0600-1800) received a single intraperitoneal injection of disopyramide (30mg per kg of body weight) at one of four different fixed time points of a 24-h period, i.e. 1000, 1600, 2200 or 0400. Blood samples were taken 0.5,1,2,3,4 and 6 hr after drug administration and total and free plasma levels of disopyramide were measured by an immunoenzymatic method.

Our data showed statistically significant circadian rhythms in the following pharmacokinetic parameters: highest volume of distribution = 3.91 ± 0.211kg^-1 at 2200 (circadian amplitude, half the peak-to-trough difference relative to the 24-hr mean multiplied by 100, is 34%); highest area under concentration curves = 16.06 ± 1.03μgml^-1hr^-1 at 0400 (circadian amplitude = 43%) and highest clearance = 3.04 ± 0.191hr“kg”¹ at 2200 (circadian amplitude = 21%). Protein binding of the drug was shown to ^he circadian time dependent. Alpha and beta phase elimination half-lives were not found to be significantly circadian phase-dependent. Thus circadian changes in disopyramide clearance may represent circadian changes in the drug's volume of distribution.     

Metabolic Phenotypes in Pancreatic Cancer

IntroductionThe aim of present study was to profile the glucose-dependent and glutamine- dependent metabolism in pancreatic cancer.MethodsWe performed Immunohistochemical staining of GLUT1, CAIX, BNIP3, p62, LC3, GLUD1, and GOT1. Based on the expression of metabolism-related proteins, the metabolic phenotypes of tumors were classified into two categories, including glucose- and glutamine-dependent metabolism. There were Warburg type, reverse Warburg type, mixed type, and null type in glucose-dependent metabolism, and canonical type, non-canonical type, mixed type, null type in glutamine-dependent metabolism.ResultsLonger overall survival was associated with high expression of BNIP3 in tumor (p = 0.010). Shorter overall survival was associated with high expression of GLUT1 in tumor (P = 0.002) and GOT1 in tumor (p = 0.030). Warburg type of glucose-dependent metabolism had a highest percentage of tumors with nerve infiltration (P = 0.0003), UICC stage (P = 0.0004), and activated autophagic status in tumor (P = 0.0167). Mixed type of glucose-dependent metabolism comprised the highest percentage of tumors with positive marginal status (P<0.0001), lymphatic invasion (P<0.0001), and activated autophagic status in stroma (P = 0.0002). Mixed type and Warburg type had a significant association with shorter overall survival (P = 0.018). Non-canonical type and mixed type of glutamine-dependent metabolism comprised the highest percentage of tumors with vascular invasion (p = 0.0073), highest percentage of activated autophagy in tumors (P = 0.0034). Moreover, these two types of glutamine-dependent metabolism were significantly associated with shorter overall survival (P<0.001). Further analysis suggested that most of tumors were dependent on both glucose- and glutamine-dependent metabolism. After dividing the tumors according to the number of metabolism, we found that the increasing numbers of metabolism subtypes inversely associated with survival outcome.ConclusionWarburg type, non-canonical type and mixed types of glucose- and glutamine-dependent metabolism comprised of more metabolically active, biologically aggressive and poor prognostic tumors. Moreover, the increasing subtypes and categories of the metabolism in each tumor significantly associated with poor prognosis.     

Oncogenic Pathway Combinations Predict Clinical Prognosis in Gastric Cancer

Many solid cancers are known to exhibit a high degree of heterogeneity in their deregulation of different oncogenic pathways. We sought to identify major oncogenic pathways in gastric cancer (GC) with significant relationships to patient survival. Using gene expression signatures, we devised an in silico strategy to map patterns of oncogenic pathway activation in 301 primary gastric cancers, the second highest cause of global cancer mortality. We identified three oncogenic pathways (proliferation/stem cell, NF-κB, and Wnt/β-catenin) deregulated in the majority (>70%) of gastric cancers. We functionally validated these pathway predictions in a panel of gastric cancer cell lines. Patient stratification by oncogenic pathway combinations showed reproducible and significant survival differences in multiple cohorts, suggesting that pathway interactions may play an important role in influencing disease behavior. Individual GCs can be successfully taxonomized by oncogenic pathway activity into biologically and clinically relevant subgroups. Predicting pathway activity by expression signatures thus permits the study of multiple cancer-related pathways interacting simultaneously in primary cancers, at a scale not currently achievable by other platforms.     

胰腺癌细胞株和胰腺癌组织中miRNAs的表达检测

目的:探明miRNAs在胰腺癌细胞株及组织中的表达情况,证实miRNAs的差异表达与胰腺癌的发生有相关性.方法:对胰腺癌细胞株和胰腺癌组织进行总RNA的提取,通过Real-time PCR方法检测17种miRNAs(miR-16、miR-20a、miR-21、miR-26a、miR-142-3p、miR-155、miR-210、miR-181a、miR-181b、miR-196a、miR-939、miR-223、miR-1202、miR-1207-5p、miR-1825、miR-1228、miR-1915)在胰腺癌细胞株及胰腺癌组织中的表达,分析miRNAs的表达与胰腺癌患者临床表现之间有无相关性.结果:胰腺癌细胞株和胰腺癌组织中miRNAs的表达明显较正常胰腺组织高.癌组织及癌旁组织中miRNAs表达量在不同性别的胰腺癌患者中差异不大(P＞0.05),并且miRNAs的表达与患者年龄及其血清CA19-9关系不大.结论:经筛选的miRNAs可以作为胰腺癌的诊断标志.胰腺癌组织中的miRNAs表达并不是一成不变的,而是随着肿瘤的生长而不断发生变化.