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1.  Tumor Suppressor Alterations Cooperate to Drive Aggressive Mesotheliomas with Enriched Cancer Stem Cells via a p53-miR34a-c-Met Axis 
Cancer research  2013;74(4):1261-1271.
Malignant mesothelioma (MM) is a highly aggressive, asbestos-related cancer frequently marked by mutations of both NF2 and CDKN2A. We demonstrate that germline knockout of one allele of each of these genes causes accelerated onset and progression of asbestos-induced MM compared to asbestos-exposed Nf2+/− or wild-type (WT) mice. Ascites from some Nf2+/−;Cdkn2a+/− mice exhibited large tumor spheroids, and tail vein injections of MM cells established from these mice, but not from Nf2+/− or WT mice, produced numerous tumors in the lung, suggesting increased metastatic potential of tumor cells from Nf2+/−;Cdkn2a+/− mice. Intraperitoneal injections of MM cells derived from Nf2+/−;Cdkn2a+/− mice into SCID mice produced tumors that penetrated the diaphragm and pleural cavity and harbored an increased cancer stem cells (CSCs). MM cells from Nf2+/−;Cdkn2a+/− mice stained positively for CSC markers and formed CSC spheroids in vitro more efficiently than counterparts from WT mice. Moreover, tumor cells from Nf2+/−;Cdkn2a+/− mice showed elevated c-Met expression/activation, which was partly dependent on p53-mediated regulation of miR34a and required for tumor migration/invasiveness and maintenance of the CSC population. Collectively, these studies demonstrate in vivo that inactivation of Nf2 and Cdkn2a cooperate to drive the development of highly aggressive MMs characterized by enhanced tumor spreading capability and the presence of a CSC population associated with p53/miR34a-dependent activation of c-Met. These findings suggest that cooperativity between losses of Nf2 and Cdkn2a plays a fundamental role in driving the highly aggressive tumorigenic phenotype considered to be a hallmark of MM.
PMCID: PMC3945416  PMID: 24371224
malignant mesothelioma; tumor suppressor genes; cancer stem cells; metastasis; mouse models
2.  Merlin deficiency predicts for FAK inhibitor sensitivity: A synthetic lethal relationship 
Science translational medicine  2014;6(237):237ra68.
The goal of targeted therapy is to match a selective drug with a genetic lesion that predicts for drug sensitivity. In a diverse panel of cancer cell lines, we found that the cells most sensitive to focal adhesion kinase (FAK) inhibition are deficient in the expression of the NF2 tumor suppressor gene product, Merlin. Merlin expression is often lost in malignant pleural mesothelioma (MPM), an asbestos-induced aggressive cancer with limited treatment options. Our data demonstrate that low Merlin expression predicts for increased sensitivity of MPM cells to a FAK inhibitor, VS-4718, in vitro and in tumor xenograft models. Disruption of MPM cell-cell or cell-extracellular matrix (ECM) contacts with blocking antibodies suggests that weak cell-cell adhesions in Merlin-negative MPM cells lead to their greater dependence on cell-ECM-induced FAK signaling. This provides one explanation of why Merlin-negative cells are vulnerable to FAK inhibitor treatment. Furthermore, we validated ALDH as a marker of cancer stem cells (CSCs) in MPM, a cell population thought to mediate tumor relapse after chemotherapy. Whereas pemetrexed and cisplatin, standard-of-care agents for MPM, enrich for CSCs, FAK inhibitor treatment preferentially eliminates these cells. These preclinical results provide the rationale for a clinical trial in MPM patients using a FAK inhibitor as a single agent after first-line chemotherapy. With this design, the FAK inhibitor could potentially induce a more durable clinical response due to reduction of CSCs along with a strong antitumor effect. Furthermore, our data suggest that patients with Merlin-negative tumors may especially benefit from FAK inhibitor treatment.
PMCID: PMC4165339  PMID: 24848258
3.  Germline Mutations in Mtap Cooperate with Myc to Accelerate Tumorigenesis in Mice 
PLoS ONE  2013;8(6):e67635.
The gene encoding the methionine salvage pathway methylthioadenosine phosphorylase (MTAP) is a tumor suppressor gene that is frequently inactivated in a wide variety of human cancers. In this study, we have examined if heterozygosity for a null mutation in Mtap (MtaplacZ) could accelerate tumorigenesis development in two different mouse cancer models, Eμ-myc transgenic and Pten+/−.
Mtap Eμ-myc and Mtap Pten mice were generated and tumor-free survival was monitored over time. Tumors were also examined for a variety of histological and protein markers. In addition, microarray analysis was performed on the livers of MtaplacZ/+ and Mtap+/+ mice.
Survival in both models was significantly decreased in MtaplacZ/+ compared to Mtap+/+ mice. In Eµ-myc mice, Mtap mutations accelerated the formation of lymphomas from cells in the early pre-B stage, and these tumors tended to be of higher grade and have higher expression levels of ornithine decarboxylase compared to those observed in control Eµ-myc Mtap+/+ mice. Surprisingly, examination of Mtap status in lymphomas in Eµ-myc MtaplacZ/+ and Eµ-myc Mtap+/+ animals did not reveal significant differences in the frequency of loss of Mtap protein expression, despite having shorter latency times, suggesting that haploinsufficiency of Mtap may be playing a direct role in accelerating tumorigenesis. Consistent with this idea, microarray analysis on liver tissue from age and sex matched Mtap+/+ and MtaplacZ/+ animals found 363 transcripts whose expression changed at least 1.5-fold (P<0.01). Functional categorization of these genes reveals enrichments in several pathways involved in growth control and cancer.
Our findings show that germline inactivation of a single Mtap allele alters gene expression and enhances lymphomagenesis in Eµ-myc mice.
PMCID: PMC3694069  PMID: 23840755
4.  Chemical Genetic Screening for Compounds that Preferentially Inhibit Growth of Methylthioadenosine Phosphorylase (MTAP) Deficient Saccharomyces Cerevisiae 
Methylthioadenosine phosphorylase (MTAP), a key enzyme in the methionine salvage pathway, is inactivated in a variety of human cancers. Since all human tissues express MTAP, it would be of potential interest to identify compounds that selectively inhibit the growth of MTAP deficient cells. To determine if MTAP inactivation could be targeted, we have performed a differential chemical genetic screen in isogenic MTAP+ and MTAP− S. cerevisiae. A low molecular weight compound library containing 30,080 unique compounds was screened for those that selectively inhibit growth of MTAP− yeast using a differential growth assay. One compound, containing a 1,3,4-thiadiazine ring, repeatedly showed a differential dose response, with MTAP− cells exhibiting a four-fold shift in IC50 compared to MTAP+ cells. Several structurally related derivatives of this compound also showed enhanced growth inhibition in MTAP− yeast. These compounds were also examined for growth inhibition of isogenic MTAP+ and MTAP− HT1080 fibrosarcoma cells, and four of the five compounds exhibited evidence of modest, but significant, increased potency in MTAP− cells. In summary, these studies show the feasibility of differential growth screening technology and have identified a novel class of compounds that can preferentially inhibit growth of MTAP− cells.
PMCID: PMC3019245  PMID: 21131597
Methionine Salvage Pathway; Drug screening; Yeast; Genetic-chemical interaction
5.  Mice Heterozygous for Germline Mutations in Methylthioadenosine Phosphorylase (MTAP) Die Prematurely of T-cell Lymphoma 
Cancer research  2009;69(14):5961-5969.
Large homozygous deletions of 9p21 that inactivate CDKN2A, ARF, and MTAP are common in a wide variety of human cancers. The role for CDKN2A and ARF in tumorigenesis is well established, but whether MTAP loss directly affects tumorigenesis is unclear. MTAP encodes the enzyme methylthioadenosine phosphorylase, a key enzyme in the methionine salvage pathway. To determine if loss of MTAP plays a functional role in tumorigenesis, we have created an MTAP-knockout mouse. Mice homozygous for a MTAP null allele (MtaplacZ) have an embryonic lethal phenotype dying around day 8 post-conception. Mtap/MtaplacZ heterozygotes are born at Mendelian frequencies and appear indistinguishable from wild-type mice during the first year of life, but they tend to die prematurely with a median survival of 585 days. Autopsies on these animals reveal that they have greatly enlarged spleens, altered thymic histology, and lymphocytic infiltration of their livers, consistent with lymphoma. Immunohistochemical staining and FACS analysis indicate that these lymphomas are primarily T-cell in origin. Lymphoma infiltrated tissues tend to have reduced levels of Mtap mRNA and MTAP protein, and unaltered levels of methyldeoxycytidine. These studies show that Mtap is a tumor suppressor gene independent of CDKN2A and ARF.
PMCID: PMC2757012  PMID: 19567676
Cancer; Tumor Suppressor Gene; Methionine; Embryonic Lethal

Results 1-5 (5)