Growth Transformation of B Cells by Epstein-Barr Virus Requires IMPDH2 Induction and Nucleolar Hypertrophy.

The in vitro growth transformation of primary B cells by Epstein-Barr virus (EBV) is the initial step in the development of posttransplant lymphoproliferative disorder (PTLD). We performed electron microscopic analysis and immunostaining of primary B cells infected with wild-type EBV. Interestingly, the nucleolar size was increased by two days after infection. A recent study found that nucleolar hypertrophy, which is caused by the induction of the IMPDH2 gene, is required for the efficient promotion of growth in cancers. In the present study, RNA-seq revealed that the IMPDH2 gene was significantly induced by EBV and that its level peaked at day 2. Even without EBV infection, the activation of primary B cells by the CD40 ligand and interleukin-4 increased IMPDH2 expression and nucleolar hypertrophy. Using EBNA2 or LMP1 knockout viruses, we found that EBNA2 and MYC, but not LMP1, induced the IMPDH2 gene during primary infections. IMPDH2 inhibition by mycophenolic acid (MPA) blocked the growth transformation of primary B cells by EBV, leading to smaller nucleoli, nuclei, and cells. Mycophenolate mofetil (MMF), which is a prodrug of MPA that is approved for use as an immunosuppressant, was tested in a mouse xenograft model. Oral MMF significantly improved the survival of mice and reduced splenomegaly. Taken together, these results indicate that EBV induces IMPDH2 expression through EBNA2-dependent and MYC-dependent mechanisms, leading to the hypertrophy of the nucleoli, nuclei, and cells as well as efficient cell proliferation. Our results provide basic evidence that IMPDH2 induction and nucleolar enlargement are crucial for B cell transformation by EBV. In addition, the use of MMF suppresses PTLD. IMPORTANCE EBV infections cause nucleolar enlargement via the induction of IMPDH2, which are essential for B cell growth transformation by EBV. Although the significance of IMPDH2 induction and nuclear hypertrophy in the tumorigenesis of glioblastoma has been reported, EBV infection brings about the change quickly by using its transcriptional cofactor, EBNA2, and MYC. Moreover, we present here, for the novel, basic evidence that an IMPDH2 inhibitor, namely, MPA or MMF, can be used for EBV-positive posttransplant lymphoproliferative disorder (PTLD).

Estimating copy number to determine BRCA2 deletion status and to expect prognosis in localized prostate cancer.

BACKGROUND: The significance of BRCA alterations has been implicated in the development of metastatic castration-resistant prostate cancer (PC). The details of the frequency and significance of BRCA alterations in localized PC remain unknown. In this study, we investigated the frequency and clinical significance of BRCA alterations in localized PCs using an in-house next-generation sequencer (NGS) system. METHODS: DNA was extracted from formalin-fixed paraffin-embedded tissues of surgical specimens from 126 patients with clinically localized PC who underwent radical prostatectomy. The mutation information of 164 cancer genes was analyzed using the PleSSision-Rapid test. Both copy number (CN) variation and loss of heterozygosity of various genes, such as BRCA1 and BRCA2, were estimated and reported. RESULTS: Next-generation sequencer analyses revealed that the BRCA2 CN was decreased in 17 patients (13.5%) and the BRCA1 CN in six (4.8%) patients. NGS-based CN values were shown to be highly correlated with droplet digital PCR-based CN values. Tissue-specific BRCA expression investigated using the Human Protein Atlas showed that the decreased CN of BRCA2, but not BRCA1, is responsible for the decreased BRCA activity in PC. Ten of the 22 patients with decreased BRCA2 CN were presumed to have somatic heterozygous deletion. There were no observed associations between the heterozygous deletion of BRCA2 and various clinicopathological parameters. Furthermore, three of 10 patients developed biochemical recurrence within 3 months after surgery. Multivariate analyses revealed that the initial prostate-specific antigen levels and BRCA2 CN were independent factors for biochemical recurrence. CONCLUSION: Our results suggest that a decrease in BRCA2 CN may be used as a biomarker for predicting recurrence after surgery in localized PC. Early screening for somatic alterations in BRCA2 using NGS may help to broadly predict the risk of PC progression.

Depletion of R270C Mutant p53 in Osteosarcoma Attenuates Cell Growth but Does Not Prevent Invasion and Metastasis In Vivo.

Novel therapeutic targets are needed to better treat osteosarcoma, which is the most common bone malignancy. We previously developed mouse osteosarcoma cells, designated AX (accelerated bone formation) cells from bone marrow stromal cells. AX cells harbor both wild-type and mutant forms of p53 (R270C in the DNA-binding domain, which is equivalent to human R273C). In this study, we showed that mutant p53 did not suppress the transcriptional activation function of wild-type p53 in AX cells. Notably, AXT cells, which are cells derived from tumors originating from AX cells, lost wild-type p53 expression, were devoid of the intact transcription activation function, and were resistant to doxorubicin. ChIP-seq analyses revealed that this mutant form of p53 bound to chromatin in the vicinity of the transcription start sites of various genes but exhibited a different binding profile from wild-type p53. The knockout of mutant p53 in AX and AXT cells by CRISPR-Cas9 attenuated tumor growth but did not affect the invasion of these cells. In addition, depletion of mutant p53 did not prevent metastasis in vivo. Therefore, the therapeutic potency targeting R270C (equivalent to human R273C) mutant p53 is limited in osteosarcoma. However, considering the heterogeneous nature of osteosarcoma, it is important to further evaluate the biological and clinical significance of mutant p53 in various cases.

Pigment epithelium-derived factor promotes peritoneal dissemination of ovarian cancer through induction of immunosuppressive macrophages.

Peritoneal dissemination of ovarian cancer (OC) correlates with poor prognosis, but the mechanisms underlying the escape of OC cells from the intraperitoneal immune system have remained unknown. We here identify pigment epithelium-derived factor (PEDF) as a promoting factor of OC dissemination, which functions through induction of CD206+ Interleukin-10 (IL-10)-producing macrophages. High PEDF gene expression in tumors is associated with poor prognosis in OC patients. Concentrations of PEDF in ascites and serum are significantly higher in OC patients than those with more benign tumors and correlated with early recurrence of OC patients, suggesting that PEDF might serve as a prognostic biomarker. Bromodomain and extraterminal (BET) inhibitors reduce PEDF expression and limit both OC cell survival and CD206+ macrophage induction in the peritoneal cavity. Our results thus implicate PEDF as a driver of OC dissemination and identify a BET protein-PEDF-IL-10 axis as a promising therapeutic target for OC.

Conventional or Drug-Eluting Beads? Randomized Controlled Study of Chemoembolization for Hepatocellular Carcinoma: JIVROSG-1302.

Introduction: With the advent of effective systemic therapy, transarterial chemoembolization (TACE) is established as a highly effective locoregional treatment modality for carefully selected patients with hepatocellular carcinoma (HCC). This randomized controlled trial was conducted to clarify whether selective TACE with drug-eluting beads (DEB-TACE) loaded with epirubicin or selective conventional TACE (cTACE) with epirubicin-ethiodized oil might be more effective for obtaining complete response(CR) in patients with HCC. Methods: Between March 2016 and May 2019, Child-Pugh class A or B patients with unresectable HCC who were scheduled to receive selective TACE were randomly assigned at a 1:1 ratio to the DEB-TACE arm or the cTACE arm. The primary endpoint was the CR rate at 3 months, as evaluated according to the modified Response Evaluation Criteria in Solid Tumors by an independent review committee, and the secondary endpoints were the CR rate at 1 month and incidences of adverse events. Results: A total of 200 patients (DEB-TACE, 99 patients; cTACE, 101 patients) were enrolled in the study. The CR rates at 3 months and 1 month were significantly higher in the cTACE arm (75.2%, 84.2%) as compared with the DEB-TACE arm (27.6%, 35.7%). However, the frequencies of adverse events of any grade, including pyrexia (DEB-TACE vs. cTACE, 19.4% vs. 45.5%, p = 0.0001), fatigue (5.1% vs. 15.8%, p = 0.0194), malaise (11.1% vs. 25.7%, p = 0.0103), appetite loss (12.1% vs. 28.7%, p = 0.0048), abdominal pain (12.1% vs. 23.8%, p = 0.0423), increased serum bilirubin (22.2% vs. 48.5%, p = 0.0002), hypoalbuminemia (43.4% vs. 60.3%, p = 0.0154), increased serum aspartate aminotransferase (35.7% vs. 81.2%, p < 0.0001), and increased serum alanine aminotransferase (35.7% vs. 77.2%, p < 0.0001), were also significantly higher in the cTACE arm than in the DEB-TACE arm. Conclusions: Selective cTACE appeared to have higher CR rates for local tumor control as compared to selective DEB-TACE for HCC. However, the frequency of postembolization syndrome was also significantly higher in the cTACE group than in the DEB-TACE group. Thus, to achieve CR, cTACE may be selected over DEB-TACE in patients who can be expected to tolerate postembolization syndrome.

Regulatory roles of fibronectin and integrin α5 in reorganization of the actin cytoskeleton and completion of adipogenesis.

Cellular differentiation is characterized by changes in cell morphology that are largely determined by actin dynamics. We previously showed that depolymerization of the actin cytoskeleton triggers the differentiation of preadipocytes into mature adipocytes as a result of inhibition of the transcriptional coactivator activity of megakaryoblastic leukemia 1 (MKL1). The extracellular matrix (ECM) influences cell morphology via interaction with integrins, and reorganization of the ECM is associated with cell differentiation. Here we show that interaction between actin dynamics and ECM rearrangement plays a key role in adipocyte differentiation. We found that depolymerization of the actin cytoskeleton precedes disruption and degradation of fibrillar fibronectin (FN) structures at the cell surface after the induction of adipogenesis in cultured preadipocytes. A FN matrix suppressed both reorganization of the actin cytoskeleton into the pattern characteristic of adipocytes and terminal adipocyte differentiation, and these inhibitory effects were overcome by knockdown of integrin α5 (ITGα5). Peroxisome proliferator-activated receptor γ was required for down-regulation of FN during adipocyte differentiation, and MKL1 was necessary for the expression of ITGα5. Our findings suggest that cell-autonomous down-regulation of FN-ITGα5 interaction contributes to reorganization of the actin cytoskeleton and completion of adipocyte differentiation.

Molecular classification and diagnostics of upper urinary tract urothelial carcinoma.

Upper urinary tract urothelial carcinoma (UTUC) is one of the common urothelial cancers. Its molecular pathogenesis, however, is poorly understood, with no useful biomarkers available for accurate diagnosis and molecular classification. Through an integrated genetic study involving 199 UTUC samples, we delineate the landscape of genetic alterations in UTUC enabling genetic/molecular classification. According to the mutational status of TP53, MDM2, RAS, and FGFR3, UTUC is classified into five subtypes having discrete profiles of gene expression, tumor location/histology, and clinical outcome, which is largely recapitulated in an independent UTUC cohort. Sequencing of urine sediment-derived DNA has a high diagnostic value for UTUC with 82.2% sensitivity and 100% specificity. These results provide a solid basis for better diagnosis and management of UTUC.

MEK inhibition preferentially suppresses anchorage-independent growth in osteosarcoma cells and decreases tumors in vivo.

Osteosarcoma is the most common high-grade malignancy of bone, and novel therapeutic options are urgently required. Previously, we developed mouse osteosarcoma AXT cells that can proliferate both under adherent and nonadherent conditions. Based on metabolite levels, nonadherent conditions were more similar to the in vivo environment than adherent conditions. A drug screen identified MEK inhibitors, including trametinib, that preferentially decreased the viability of nonadherent AXT cells. Trametinib inhibited the cell cycle and induced apoptosis in AXT cells, and both effects were stronger under nonadherent conditions. Trametinib also potently decreased viability in U2OS cells, but its effects were less prominent in MG63 or Saos2 cells. By contrast, MG63 and Saos2 cells were more sensitive to PI3K inhibition than AXT or U2OS cells. Notably, the combination of MAPK/ERK kinase (MEK) and PI3K inhibition synergistically decreased viability in U2OS and AXT cells, but this effect was less pronounced in MG63 or Saos2 cells. Therefore, signal dependence for cell survival and crosstalk between MEK-ERK and PI3K-AKT pathways in osteosarcoma are cell context-dependent. The activation status of other kinases including CREB varied in a cell context-dependent manner, which might determine the response to MEK inhibition. A single dose of trametinib was sufficient to decrease the size of the primary tumor and circulating tumor cells in vivo. Moreover, combined administration of trametinib and rapamycin or conventional anticancer drugs further increased antitumor activity. Thus, given optimal biomarkers for predicting its effects, trametinib holds therapeutic potential for the treatment of osteosarcoma.

Oncogenic KRAS-expressing organoids with biliary epithelial stem cell properties give rise to biliary tract cancer in mice.

Biliary tract cancer (BTC) arises from biliary epithelial cells (BECs) and includes intrahepatic cholangiocarcinoma (IHCC), gallbladder cancer (GC), and extrahepatic cholangiocarcinoma (EHCC). Although frequent KRAS mutations and epigenetic changes at the INK4A/ARF locus have been identified, the molecular pathogenesis of BTC is unclear and the development of corresponding anticancer agents remains inadequate. We isolated epithelial cell adhesion molecule (EpCAM)-positive BECs from the mouse intrahepatic bile duct, gallbladder, and extrahepatic bile duct, and established organoids derived from these cells. Introduction of activated KRAS and homozygous deletion of Ink4a/Arf in the cells of each organoid type conferred the ability to form lethal metastatic adenocarcinoma with differentiated components and a pronounced desmoplastic reaction on cell transplantation into syngeneic mice, indicating that the manipulated cells correspond to BTC-initiating cells. The syngeneic mouse models recapitulate the pathological features of human IHCC, GC, and EHCC, and they should therefore prove useful for the investigation of BTC carcinogenesis and the development of new therapeutic strategies. Tumor cells isolated from primary tumors formed organoids in three-dimensional culture, and serial syngeneic transplantation of these cells revealed that their cancer stem cell properties were supported by organoid culture, but not by adherent culture. Adherent culture thus attenuated tumorigenic activity as well as the expression of both epithelial and stem cell markers, whereas the expression of epithelial-mesenchymal transition (EMT)-related transcription factor genes and mesenchymal cell markers was induced. Our data show that organoid culture is important for maintenance of epithelial cell characteristics, stemness, and tumorigenic activity of BTC-initiating cells.

The Inhibitor of Apoptosis Protein Livin Confers Resistance to Fas-Mediated Immune Cytotoxicity in Refractory Lymphoma.

Death receptor Fas-mediated apoptosis not only eliminates nonspecific and autoreactive B cells but also plays a major role in antitumor immunity. However, the possible mechanisms underlying impairment of Fas-mediated induction of apoptosis during lymphomagenesis remain unknown. In this study, we employed our developed syngeneic lymphoma model to demonstrate that downregulation of Fas is required for both lymphoma development and lymphoma cell survival to evade immune cytotoxicity. CD40 signal activation significantly restored Fas expression and thereby induced apoptosis after Fas ligand treatment in both mouse and human lymphoma cells. Nevertheless, certain human lymphoma cell lines were found to be resistant to Fas-mediated apoptosis, with Livin (melanoma inhibitor of apoptosis protein; ML-IAP) identified as a driver of such resistance. High expression of Livin and low expression of Fas were associated with poor prognosis in patients with aggressive non-Hodgkin's lymphoma. Livin expression was tightly driven by bromodomain and extraterminal (BET) proteins BRD4 and BRD2, suggesting that Livin expression is epigenetically regulated in refractory lymphoma cells to protect them from Fas-mediated apoptosis. Accordingly, the combination of CD40-mediated Fas restoration with targeting of the BET proteins-Livin axis may serve as a promising immunotherapeutic strategy for refractory B-cell lymphoma. SIGNIFICANCE: These findings yield insights into identifying risk factors in refractory lymphoma and provide a promising therapy for tumors resistant to Fas-mediated antitumor immunity. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/20/4439/F1.large.jpg.

Combined Cohesin-RUNX1 Deficiency Synergistically Perturbs Chromatin Looping and Causes Myelodysplastic Syndromes.

STAG2 encodes a cohesin component and is frequently mutated in myeloid neoplasms, showing highly significant comutation patterns with other drivers, including RUNX1. However, the molecular basis of cohesin-mutated leukemogenesis remains poorly understood. Here we show a critical role of an interplay between STAG2 and RUNX1 in the regulation of enhancer-promoter looping and transcription in hematopoiesis. Combined loss of STAG2 and RUNX1, which colocalize at enhancer-rich, CTCF-deficient sites, synergistically attenuates enhancer-promoter loops, particularly at sites enriched for RNA polymerase II and Mediator, and deregulates gene expression, leading to myeloid-skewed expansion of hematopoietic stem/progenitor cells (HSPC) and myelodysplastic syndromes (MDS) in mice. Attenuated enhancer-promoter loops in STAG2/RUNX1-deficient cells are associated with downregulation of genes with high basal transcriptional pausing, which are important for regulation of HSPCs. Downregulation of high-pausing genes is also confirmed in STAG2-cohesin-mutated primary leukemia samples. Our results highlight a unique STAG2-RUNX1 interplay in gene regulation and provide insights into cohesin-mutated leukemogenesis. SIGNIFICANCE: We demonstrate a critical role of an interplay between STAG2 and a master transcription factor of hematopoiesis, RUNX1, in MDS development, and further reveal their contribution to regulation of high-order chromatin structures, particularly enhancer-promoter looping, and the link between transcriptional pausing and selective gene dysregulation caused by cohesin deficiency.This article is highlighted in the In This Issue feature, p. 747.

Frequent mutations that converge on the NFKBIZ pathway in ulcerative colitis.

Chronic inflammation is accompanied by recurring cycles of tissue destruction and repair and is associated with an increased risk of cancer1-3. However, how such cycles affect the clonal composition of tissues, particularly in terms of cancer development, remains unknown. Here we show that in patients with ulcerative colitis, the inflamed intestine undergoes widespread remodelling by pervasive clones, many of which are positively selected by acquiring mutations that commonly involve the NFKBIZ, TRAF3IP2, ZC3H12A, PIGR and HNRNPF genes and are implicated in the downregulation of IL-17 and other pro-inflammatory signals. Mutational profiles vary substantially between colitis-associated cancer and non-dysplastic tissues in ulcerative colitis, which indicates that there are distinct mechanisms of positive selection in both tissues. In particular, mutations in NFKBIZ are highly prevalent in the epithelium of patients with ulcerative colitis but rarely found in both sporadic and colitis-associated cancer, indicating that NFKBIZ-mutant cells are selected against during colorectal carcinogenesis. In further support of this negative selection, we found that tumour formation was significantly attenuated in Nfkbiz-mutant mice and cell competition was compromised by disruption of NFKBIZ in human colorectal cancer cells. Our results highlight common and discrete mechanisms of clonal selection in inflammatory tissues, which reveal unexpected cancer vulnerabilities that could potentially be exploited for therapeutics in colorectal cancer.

ROCK Inhibition Induces Terminal Adipocyte Differentiation and Suppresses Tumorigenesis in Chemoresistant Osteosarcoma Cells.

Tumors comprise heterogeneous cell types including cancer stem cells (CSC), progenitor cells, and differentiated cells. Chemoresistance is a potential cause of relapse and a key characteristic of CSC, but the development of novel therapeutic approaches for targeting these cells has been limited. We previously established osteosarcoma-initiating (OSi) cells by introducing the gene for c-Myc into bone marrow stromal cells of Ink4a/Arf knockout mice. These OSi cells are composed of two distinct clones: highly tumorigenic cells (AX cells), similar to bipotent committed osteochondral progenitor cells, and tripotent cells of low tumorigenicity (AO cells), similar to mesenchymal stem cells. Here we show that depolymerization of the actin cytoskeleton induces terminal adipocyte differentiation and suppresses tumorigenesis in chemoresistant OSi cells. In contrast to AX cells, AO cells were highly resistant to conventional chemotherapeutic agents such as doxorubicin and were thus identified as chemoresistant cells. Inhibition of Rho-associated coiled-coil containing protein kinase (ROCK) elicited terminal adipocyte differentiation in chemoresistant AO cells through negative regulation of the transcriptional coactivator megakaryoblastic leukemia 1 associated with actin depolymerization. The clinically administered ROCK inhibitor fasudil significantly suppressed growth in vitro and tumorigenicity in vivo of chemoresistant AO cells as well as of OSi cells. Our findings thus suggest a new therapeutic strategy based on the induction of trans-terminal differentiation via modulation of actin cytoskeleton dynamics for therapy-resistant osteosarcoma stem cells. SIGNIFICANCE: These findings suggest that induction of trans-terminal differentiation through regulation of actin dynamics is a potential novel therapeutic approach for targeting chemoresistant stem-like tumor cells.

T315I mutation of BCR-ABL1 into human Philadelphia chromosome-positive leukemia cell lines by homologous recombination using the CRISPR/Cas9 system.

In many cancers, somatic mutations confer tumorigenesis and drug-resistance. The recently established clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a potentially elegant approach to functionally evaluate mutations in cancers. To reproduce mutations by homologous recombination (HR), the HR pathway must be functional, but DNA damage repair is frequently impaired in cancers. Imatinib is a tyrosine kinase inhibitor for BCR-ABL1 in Philadelphia chromosome-positive (Ph+) leukemia, and development of resistance due to kinase domain mutation is an important issue. We attempted to introduce the T315I gatekeeper mutation into three Ph+ myeloid leukemia cell lines with a seemingly functional HR pathway due to resistance to the inhibitor for poly (ADP) ribose polymerase1. Imatinib-resistant sublines were efficiently developed by the CRISPR/Cas9 system after short-term selection with imatinib; resulting sublines acquired the T315I mutation after HR. Thus, the usefulness of CRISPR/Cas9 system for functional analysis of somatic mutations in cancers was demonstrated.

Periostin antisense oligonucleotide suppresses bleomycin-induced formation of a lung premetastatic niche for melanoma.

Metastasis is the leading cause of cancer death. A tumor-supportive microenvironment, or premetastatic niche, at potential secondary tumor sites plays an important role in metastasis, especially in tumor cell colonization. Although a fibrotic milieu is known to promote tumorigenesis and metastasis, the underlying molecular contributors to this effect have remained unclear. Here we show that periostin, a component of the extracellular matrix that functions in tissue remodeling, has a key role in formation of a fibrotic environment that promotes tumor metastatic colonization. We found that periostin was widely expressed in fibrotic lesions of mice with bleomycin-induced lung fibrosis, and that up-regulation of periostin expression coincided with activation of myofibroblasts positive for α-smooth muscle actin. We established a lung metastasis model for B16 murine melanoma cells and showed that metastatic colonization of the lung by these cells was markedly promoted by bleomycin-induced lung fibrosis. Inhibition of periostin expression by giving an intratracheal antisense oligonucleotide targeting periostin mRNA was found to suppress bleomycin-induced lung fibrosis and thereby to attenuate metastatic colonization of the lung by melanoma cells. Our results indicate that periostin is a key player in the development of bleomycin-induced fibrosis and consequent enhancement of tumor cell colonization in the lung. Our results therefore implicate periostin as a potential target for prevention or treatment of lung metastasis.

Clofarabine exerts antileukemic activity against cytarabine-resistant B-cell precursor acute lymphoblastic leukemia with low deoxycytidine kinase expression.

Cytosine arabinoside (Ara-C) is one of the key drugs for the treatment of acute myeloid leukemia. It is also used for consolidation therapy of acute lymphoblastic leukemia (ALL). Ara-C is a deoxyadenosine analog and is phosphorylated to form cytosine arabinoside triphosphate (Ara-CTP) as an active form. In the first step of the metabolic pathway, Ara-C is phosphorylated to Ara-CMP by deoxycytidine kinase (DCK). However, the current cumulative evidence in the association of the Ara-C sensitivity in ALL appears inconclusive. We analyzed various cell lines for the possible involvement of DCK in the sensitivities of B-cell precursor ALL (BCP-ALL) to Ara-C. Higher DCK expression was associated with higher Ara-C sensitivity. DCK knockout by genome editing with a CRISPR-Cas9 system in an Ara-C-sensitive-ALL cell line induced marked resistance to Ara-C, but not to vincristine and daunorubicin, indicating the involvement of DCK expression in the Ara-C sensitivity of BCP-ALL. DCK gene silencing due to the hypermethylation of a CpG island and reduced DCK activity due to a nonsynonymous variant allele were not associated with Ara-C sensitivity. Clofarabine is a second-generation deoxyadenosine analog rationally synthesized to improve stability and reduce toxicity. The IC50 of clofarabine in 79 BCP-ALL cell lines was approximately 20 times lower than that of Ara-C. In contrast to Ara-C, although the knockout of DCK induced marked resistance to clofarabine, sensitivity to clofarabine was only marginally associated with DCK gene expression level, suggesting a possible efficacy of clofarabine for BCP-ALL that shows relative Ara-C resistance due to low DCK expression.

Development of a functional thyroid model based on an organoid culture system.

The low turnover rate of thyroid follicular cells and the lack of a long-term thyroid cell culture system have hampered studies of thyroid carcinogenesis. We have now established a thyroid organoid culture system that supports thyroid cell proliferation in vitro. The established mouse thyroid organoids performed thyroid functions including thyroglobulin synthesis, iodide uptake, and the production and release of thyroid hormone. Furthermore, transplantation of the organoids into recipient mice resulted in the formation of normal thyroid-like tissue capable of iodide uptake and thyroglobulin production in vivo. Finally, forced expression of oncogenic NRAS (NRASQ61R) in thyroid organoids established from p53 knockout mice and transplantation of the manipulated organoids into mouse recipients generated a model of poorly differentiated thyroid cancer. Our findings suggest that this newly developed thyroid organoid culture system is a potential research tool for the study of thyroid physiology and pathology including thyroid cancer.

FZR1 loss increases sensitivity to DNA damage and consequently promotes murine and human B-cell acute leukemia.

FZR1 (fizzy-related protein homolog; also known as CDH1 [cell division cycle 20 related 1]) functions in the cell cycle as a specific activator of anaphase-promoting complex or cyclosome ubiquitin ligase, regulating late mitosis, G1 phase, and activation of the G2-M checkpoint. FZR1 has been implicated as both a tumor suppressor and oncoprotein, and its precise contribution to carcinogenesis remains unclear. Here, we examined the role of FZR1 in tumorigenesis and cancer therapy by analyzing tumor models and patient specimens. In an Fzr1 gene-trap mouse model of B-cell acute lymphoblastic leukemia (B-ALL), mice with Fzr1-deficient B-ALL survived longer than those with Fzr1-intact disease, and sensitivity of Fzr1-deficient B-ALL cells to DNA damage appeared increased. Consistently, conditional knockdown of FZR1 sensitized human B-ALL cell lines to DNA damage-induced cell death. Moreover, multivariate analyses of reverse-phase protein array of B-ALL specimens from newly diagnosed B-ALL patients determined that a low FZR1 protein expression level was an independent predictor of a longer remission duration. The clinical benefit of a low FZR1 expression level at diagnosis was no longer apparent in patients with relapsed B-ALL. Consistent with this result, secondary and tertiary mouse recipients of Fzr1-deficient B-ALL cells developed more progressive and radiation-resistant disease than those receiving Fzr1-intact B-ALL cells, indicating that prolonged inactivation of Fzr1 promotes the development of resistant clones. Our results suggest that reduction of FZR1 increases therapeutic sensitivity of B-ALL and that transient rather than tonic inhibition of FZR1 may be a therapeutic strategy.

Simvastatin-Induced Apoptosis in Osteosarcoma Cells: A Key Role of RhoA-AMPK/p38 MAPK Signaling in Antitumor Activity.

Osteosarcoma is the most common type of primary bone tumor, novel therapeutic agents for which are urgently needed. To identify such agents, we screened a panel of approved drugs with a mouse model of osteosarcoma. The screen identified simvastatin, which inhibited the proliferation and migration of osteosarcoma cells in vitro Simvastatin also induced apoptosis in osteosarcoma cells in a manner dependent on inhibition of the mevalonate biosynthetic pathway. It also disrupted the function of the small GTPase RhoA and induced activation of AMP-activated protein kinase (AMPK) and p38 MAPK, with AMPK functioning upstream of p38 MAPK. Inhibitors of AMPK or p38 MAPK attenuated the induction of apoptosis by simvastatin, whereas metformin enhanced this effect of simvastatin by further activation of AMPK. Although treatment with simvastatin alone did not inhibit osteosarcoma tumor growth in vivo, its combination with a fat-free diet induced a significant antitumor effect that was enhanced further by metformin administration. Our findings suggest that simvastatin induces apoptosis in osteosarcoma cells via activation of AMPK and p38 MAPK, and that, in combination with other approaches, it holds therapeutic potential for osteosarcoma. Mol Cancer Ther; 16(1); 182-92. ©2016 AACR.