TACCing on new functions for the TSC2 tumor suppressor

Lewis Katz School of MedicineCancer and Cellular Biolog

HEF1-Aurora A Interactions

Regulated timing of cell division cycles, and geometrical precision in the planar orien-tation of cell division, are critical during organismal development and remain important for the maintenance of polarized structures in adults. Mounting evidence suggests that these processes are coordinated at the centrosome through the action of proteins that mediate both cell cycle and cell attachment. Our recent work identifying HEF1 as anactivator of the Aurora A kinase suggests a novel hub for such integrated signaling. Wesuggest that defects in components of the machinery specifying the temporal and spatial integration of cell division may induce cancer and other diseases through pleiotropic effects on cell migration, proliferation, apoptosis, and genomic stability.Lewis Katz School of MedicineCancer and Cellular Biolog

A Synthetic Lethality Screen Using a Focused siRNA Library to Identify Sensitizers to Dasatinib Therapy for the Treatment of Epithelial Ovarian Cancer.

Molecular targeted therapies have been the focus of recent clinical trials for the treatment of patients with recurrent epithelial ovarian cancer (EOC). The majority have not fared well as monotherapies for improving survival of these patients. Poor bioavailability, lack of predictive biomarkers, and the presence of multiple survival pathways can all diminish the success of a targeted agent. Dasatinib is a tyrosine kinase inhibitor of the Src-family kinases (SFK) and in preclinical studies shown to have substantial activity in EOC. However, when evaluated in a phase 2 clinical trial for patients with recurrent or persistent EOC, it was found to have minimal activity. We hypothesized that synthetic lethality screens performed using a cogently designed siRNA library would identify second-site molecular targets that could synergize with SFK inhibition and improve dasatinib efficacy. Using a systematic approach, we performed primary siRNA screening using a library focused on 638 genes corresponding to a network centered on EGFR, HER2, and the SFK-scaffolding proteins BCAR1, NEDD9, and EFS to screen EOC cells in combination with dasatinib. We followed up with validation studies including deconvolution screening, quantitative PCR to confirm effective gene silencing, correlation of gene expression with dasatinib sensitivity, and assessment of the clinical relevance of hits using TCGA ovarian cancer data. A refined list of five candidates (CSNK2A1, DAG1, GRB2, PRKCE, and VAV1) was identified as showing the greatest potential for improving sensitivity to dasatinib in EOC. Of these, CSNK2A1, which codes for the catalytic alpha subunit of protein kinase CK2, was selected for additional evaluation. Synergistic activity of the clinically relevant inhibitor of CK2, CX-4945, with dasatinib in reducing cell proliferation and increasing apoptosis was observed across multiple EOC cell lines. This overall approach to improving drug efficacy can be applied to other targeted agents that have similarly shown poor clinical activity

Alignment of U3 region sequences of mammalian type C viruses: identification of highly conserved motifs and implications for enhancer design

We aligned published sequences for the U3 region of 35 type C mammalian retroviruses. The alignment reveals that certain sequence motifs within the U3 region are strikingly conserved. A number of these motifs correspond to previously identified sites. In particular, we found that the enhancer region of most of the viruses examined contains a binding site for leukemia virus factor b, a viral corelike element, the consensus motif for nuclear factor 1, and the glucocorticoid response element. Most viruses containing more than one copy of enhancer sequences include these binding sites in both copies of the repeat. We consider this set of binding sites to constitute a framework for the enhancers of this set of viruses. Other highly conserved motifs in the U3 region include the retrovirus inverted repeat sequence, a negative regulatory element, and the CCAAT and TATA boxes. In addition, we identified two novel motifs in the promoter region that were exceptionally highly conserved but have not been previously described.Lewis Katz School of MedicineCancer and Cellular Biolog

Disease specificity of nondefective Friend and Moloney murine leukemia viruses is controlled by a small number of nucleotides

Moloney murine leukemia virus induces T cell lymphomas after injection into NFS mice, whereas the nondefective Friend virus induces erythroleukemias. Previous studies showed that sequences encompassing the viral transcriptional signals in U3 are the primary determinant of this phenotype in recombinants between these two viruses. To more precisely identify the sequences responsible, we constructed additional recombinants, within U3, between Friend and Moloney viruses and assayed these recombinants for for their disease specificity. We found that a fragment 191 bases long that included the direct repeat (enhancer) region plus 22 nucleotides to its 3' side from Friend virus was sufficient to convert Moloney virus to a virus that induced only erythroleukemias. A 171-base-long fragment of Moloney virus, including just the direct repeat, converted Friend virus to a virus that induced primarily lymphomas (about 85% of mice injected). We also constructed Moloney and Friend virus variants with one rather than two copies of the enhancer element. These viruses retained their disease specificity, although they exhibited a marked increase in the latent period of disease induction. Together the results suggest that 25 or fewer nucleotide differences, lying within and also just 3' of the direct repeat, are the primary determinant of the distinct disease specificities of nondefective Friend and Moloney viruses.Lewis Katz School of MedicineCancer and Cellular Biolog

Human RNA polymerase II subunit hsRPB7 functions in yeast and influences stress survival and cell morphology

Using a screen to identify human genes that promote pseudohyphal conversion in Saccharomyces cerevisiae, we obtained a cDNA encoding hsRPB7, a human homologue of the seventh largest subunit of yeast RNA polymerase II (RPB7). Overexpression of yeast RPB7 in a comparable strain background caused more pronounced cell elongation than overexpression of hsRPB7. hsRPB7 sequence and function are strongly conserved with its yeast counterpart because its expression can rescue deletion of the essential RPB7 gene at moderate temperatures. Further, immuno-precipitation of RNA polymerase II from yeast cells containing hsRPB7 revealed that the hsRPB7 assembles the complete set of 11 other yeast subunits. However, at temperature extremes and during maintenance at stationary phase, hsRPB7-containing yeast cells lose viability rapidly, stress-sensitive phenotypes reminiscent of those associated with deletion of the RPB4 subunit with which RPB7 normally complexes. Two-hybrid analysis revealed that although hsRPB7 and RPB4 interact, the association is of lower affinity than the RPB4-RPB7 interaction, providing a probable mechanism for the failure of hsRPB7 to fully function in yeast cells at high and low temperatures. Finally, surprisingly, hsRPB7 RNA in human cells is expressed in a tissue-specific pattern that differs from that of the RNA polymerase II largest subunit, implying a potential regulatory role for hsRPB7. Taken together, these results suggest that some RPB7 functions may be analogous to those possessed by the stress-specific prokaryotic sigma factor rpoS.Lewis Katz School of MedicineCancer and Cellular Biolog

Distinct segments within the enhancer region collaborate to specify the type of leukemia induced by nondefective Friend and Moloney viruses

The nondefective Moloney and Friend murine leukemia viruses induce T-cell lymphomas and erythroleukemias, respectively, after being injected into newborn NFS mice. In previous studies, we showed that the distinct disease specificities of the two viruses could be switched by exchanging a small segment, about 200 nucleotides in length, encompassing their enhancer regions. This segment included the direct repeat sequence and an adjacent GC-rich region of about 20 nucleotides defined in studies of Moloney murine sarcoma virus enhancer-promoter function (L. A. Laimins, P. Gruss, R. Pozzatti, and G. Khoury, J. Virol. 49:183-189, 1984). The direct repeats of Friend and Moloney viruses are identical in a central core sequence of 32 nucleotides but have sequence differences on either side of this core as well as in their GC-rich segments. To determine whether disease specificity resides in part or in all of the direct repeat and GC-rich region, we constructed recombinants between Friend and Moloney viruses within this segment and tested them for their disease-inducing phenotypes. We found that disease specificity, in particular the ability of Friend virus sequence to confer erythroleukemogenicity on Moloney virus, is encoded throughout the region in at least three separable segments: the 5' and 3' halves of the direct repeat and the GC-rich segment. When just one of these segments (either both 5' halves of the direct repeat, both 3' halves, or just the GC-rich segment) from Friend virus was substituted into a Moloney virus genome, it conferred only a negligible or low incidence of erythroleukemia (less than or equal to 5% to between 10 and 15%). Any two segments together were considerably more potent (35 to 95% erythroleukemia), with the most effective pair being the two halves of the direct repeat. Individual segments and pairs of segments were considerably more potent determinants when they were matched with a genome of the same origin. Thus, although sequences outside the enhancer region are minor determinants of disease specificity when the enhancer is derived entirely from either Friend or Moloney virus, they can play a significant role when the enhancer is of mixed origin. Some recombinant enhancers conferred a long latent period of disease induction. This was particularly striking when the 5' halves of each copy of the direct repeat sequence were derived from Moloney virus and the 3' halves were derived from Friend virus.(ABSTRACT TRUNCATED AT 400 WORDS)Lewis Katz School of MedicineCancer and Cellular Biolog

Mutation of the core or adjacent LVb elements of the Moloney murine leukemia virus enhancer alters disease specificity

Transcriptional enhancers of replication-competent mouse C-type retroviruses are potent determinants of the distinct disease-inducing phenotypes of different viral isolates and can also strongly influence the incidence and latent period of disease induction. To study the contribution of individual protein-binding sites to viral pathogenicity, we introduced mutations into each of the known nuclear factor-binding sites in the enhancer region of the Moloney murine leukemia virus and injected viruses with these mutations into newborn NFS mice. All viruses induced disease. Viruses with mutations in both copies of the leukemia virus factor a (LVa) site, leukemia virus factor c (LVc) site, or in just the promoter proximal copy of the glucocorticoid response element (GRE) had a latent period of disease onset and disease specificity indistinguishable from that of the wild-type Moloney virus. Viruses with mutations in two or three of the GREs, in both copies of the leukemia virus factor b (LVb) site, in two of the four nuclear factor 1 (NF1) consensus motifs, or in both copies of the conserved viral core element showed a significant delay in latent period of disease induction. Strikingly, viruses with mutations in the core element induced primarily erythroleukemias, and mutations in the LVb site also resulted in a significant incidence of erythroleukemias. These and other genetic and biochemical studies suggest models for how subtle alterations in the highly conserved structure of mouse C-type retrovirus enhancers can produce a dramatic effect on disease specificity.Lewis Katz School of MedicineCancer and Cellular Biolog

The Tuberous Sclerosis 2 Gene Product, Tuberin, Functions as a Rab5 GTPase Activating Protein (GAP) in Modulating Endocytosis

The tuberous sclerosis complex 2 (TSC2) is a tumor suppressor gene that plays a causative role in the autosomal dominant syndrome of tuberous sclerosis. The latter is characterized by the development of hamartomas and occasional malignancies. Expression of the wild-type gene in TSC2 mutant tumor cells inhibits proliferation and tumorigenicity. This “suppressor− activity is encoded by functional domain(s) in the C terminus that contains homology to Rap1GAP. Using a yeast two-hybrid assay to identify proteins that interact with the C-terminal domain of tuberin, the product of TSC2, a cytosolic factor, rabaptin-5, was found to associate with a distinct domain lying adjacent to the TSC2 GAP homology region. Rabaptin-5 also binds the active form of GTPase Rab5. Immune complexes of native tuberin, as well as recombinant protein, possessed activity to stimulate GTP hydrolysis of Rab5. Tuberin GAP activity was specific for Rab5 and showed no cross-reactivity with Rab3a or Rab6. Cells lacking tuberin possessed minimal Rab5GAP activity and were associated with an increased uptake of horseradish peroxidase. Re-expression of tuberin in TSC2 mutant cells reduced the rate of fluid-phase endocytosis. These findings suggest that tuberin functions as a Rab5GAP in vivo to negatively regulate Rab5-GTP activity in endocytosis.Lewis Katz School of MedicineCancer and Cellular Biolog

DUSP6 regulates drug sensitivity by modulating DNA damage response

Background: Dual specificity phosphatase 6 (DUSP6) is a member of a family of mitogen-activated protein kinase phosphatases that dephosphorylates and inhibits activated ERK1/2. Dual specificity phosphatase 6 is dynamically regulated in developmental and pathological conditions such as cancer. Methods: Cancer cell lines were made deficient in DUSP6 by siRNA and shRNA silencing. Sensitivity to anti-EGFR and chemotherapeutic agents was determined in viability and apoptosis assays, and in xenografts established in SCID mice. Cellular effects of DUSP6 inactivation were analysed by proteomic methods, followed by analysis of markers of DNA damage response (DDR) and cell cycle. Results: We determined that depletion of DUSP6 reduced the viability of cancer cell lines and increased the cytotoxicity of EGFR and other targeted inhibitors, and cytotoxic agents, in vitro and in vivo. Subsequent phosphoproteomic analysis indicated DUSP6 depletion significantly activated CHEK2 and p38, which function in the DDR pathway, and elevated levels of phosphorylated H2AX, ATM, and CHEK2, for the first time identifying a role for DUSP6 in regulating DDR. Conclusion: Our results provide a novel insight into the DUSP6 function in regulating genomic integrity and sensitivity to chemotherapy in cancer.Lewis Katz School of MedicineCancer and Cellular Biolog