Recent Scientific Papers

For more papers, visit a faculty member's page from the listing on Whitehead Faculty and access the PubMed link.

The Rag Gtpases Bind Raptor and Mediate Amino Acid Signaling to Mtorc1.
Science. 2008 May 22. [Epub ahead of print]
Sancak, Y.*, Peterson, T.R.*, Shaul, Y.D.*, Lindquist, R.A.*, Thoreen, C.C.*, Bar-Peled, L.*, and Sabatini, D.M.*
The multiprotein mTORC1 protein kinase complex is the central component of a pathway that promotes growth in response to insulin, energy levels, and amino acids, and is deregulated in common cancers. We find that the Rag proteins-a family of four related small guanosine triphosphatases (GTPases)-interact with mTORC1 in an amino acid-sensitive manner and are necessary for the activation of the mTORC1 pathway by amino acids. A Rag mutant that is constitutively bound to GTP interacted strongly with mTORC1, and its expression within cells made the mTORC1 pathway resistant to amino acid deprivation. Conversely, expression of a GDP-bound Rag mutant prevented stimulation of mTORC1 by amino acids. The Rag proteins do not directly stimulate the kinase activity of mTORC1, but, like amino acids, promote the intracellular localization of mTOR to a compartment that also contains its activator Rheb.

Coevolution in the Tumor Microenvironment.
Nature Genetics. 2008 May;40(5):494-5.
Weinberg, R.A.*
The progression of carcinomas to high- grade malignancies is accompanied by profound histological changes in the tumor-associated stroma. Although previous studies have suggested that mesenchymal cells of the stroma undergo genetic alterations during this progression, a new study now provides evidence that strongly contradicts this theory of stromal cell coevolution.

Discovery of Quinolinediones Exhibiting a Heat Shock Response and Angiogenesis Inhibition.
J Med Chem. 2008 Apr 24;51(8):2492-501. Epub 2008 Mar 26.
Hargreaves, R.H.J., David, C.L., Whitesell, L.J.*, LaBarbera, D.V., Jamil, A., Chapuis, J.C., and Skibo, E.B.
A series of substituted quinoline-5,8-diones were synthesized and evaluated as inhibitors of the chaperone protein Hsp90 using two assays: competition for binding to C-terminal ATP-binding site and competition for binding to N-terminal ATP-binding site. In addition, the ability of the compounds to induce the heat shock response was determined using a reporter fibroblast cell line. Of all the compounds assayed, only 6-aziridinyl-2-biphenylquinoline-5,8-dione induced a heat shock response and did so without interacting at the ATP binding sites of Hsp90. COMPARE analysis was carried out on quinoline-5,8-diones active in the National Cancer Institute's 60-cell line screen with the goal of discovering quinoline-5,8-dione structures that interact with other cellular targets (molecular targets) important for cancer chemotherapy. COMPARE analysis led to the discovery of a combretastatin- like quinoline-5,8-dione structure that, in fact, inhibited angiogenesis.

Detection of Compounds That Rescue Rab1-Synuclein Toxicity.
Methods Enzymol. 2008;439:339-51.
Fleming, J., Outeiro, T.F.*, Slack, M., Lindquist, S.L.*, and Bulawa, C.E.
Recent studies implicate a disruption in Rab-mediated protein trafficking as a possible contributing factor to neurodegeneration in Parkinson's disease (PD). Misfolding of the neuronal protein alpha-synuclein (asyn) is implicated in PD. Overexpression of asyn results in cell death in a wide variety of model systems, and in several organisms, including yeast, worms, flies, and rodent primary neurons, this toxicity is suppressed by the overproduction of Rab proteins. These and other findings suggest that asyn interferes with Rab function and provide new avenues for PD drug discovery. This chapter describes two assay formats that have been used successfully to identify small molecules that rescue asyn toxicity in yeast. The 96-well format monitors rescue by optical density and is suitable for screening thousands of compounds. A second format measures viable cells by reduction of the dye alamarBlue, a readout that is compatible with 96-, 384-, and 1536-well plates allowing the screening of large libraries (>100,000 compounds). A secondary assay to eliminate mechanistically undesirable hits is also described.

Loss of E-Cadherin Promotes Metastasis Via Multiple Downstream Transcriptional Pathways.
Cancer Res. 2008 May 15;68(10):3645-54.
Onder, T.T.*, Gupta, P.B., Mani, S.A.*, Yang, J., Lander, E.S., and Weinberg, R.A.*
Loss of the epithelial adhesion molecule E-cadherin is thought to enable metastasis by disrupting intercellular contacts-an early step in metastatic dissemination. To further investigate the molecular basis of this notion, we use two methods to inhibit E-cadherin function that distinguish between E-cadherin's cell-cell adhesion and intracellular signaling functions. Whereas the disruption of cell-cell contacts alone does not enable metastasis, the loss of E-cadherin protein does, through induction of an epithelial-to-mesenchymal transition, invasiveness, and anoikis resistance. We find the E-cadherin binding partner beta-catenin to be necessary, but not sufficient, for induction of these phenotypes. In addition, gene expression analysis shows that E-cadherin loss results in the induction of multiple transcription factors, at least one of which, Twist, is necessary for E-cadherin loss-induced metastasis. These findings indicate that E-cadherin loss in tumors contributes to metastatic dissemination by inducing wide-ranging transcriptional and functional changes.

The Epithelial-Mesenchymal Transition Generates Cells with Properties of Stem Cells.
Cell. 2008 May 16;133(4):704-15.
Mani, S.A.*, Guo, W.*, Liao, M.J.*, Eaton, E.N.*, Ayyanan, A., Zhou, A.Y.*, Brooks, M.*, Reinhard, F.*, Zhang, C.C.*, Shipitsin, M., Campbell, L.L., Polyak, K., Brisken, C., Yang, J., Weinberg, R.A.*
The epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer invasion and metastasis. We here report that the induction of an EMT in immortalized human mammary epithelial cells (HMLEs) results in the acquisition of mesenchymal traits and in the expression of stem-cell markers. Furthermore, we show that those cells have an increased ability to form mammospheres, a property associated with mammary epithelial stem cells. Independent of this, stem cell-like cells isolated from HMLE cultures form mammospheres and express markers similar to those of HMLEs that have undergone an EMT. Moreover, stem-like cells isolated either from mouse or human mammary glands or mammary carcinomas express EMT markers. Finally, transformed human mammary epithelial cells that have undergone an EMT form mammospheres, soft agar colonies, and tumors more efficiently. These findings illustrate a direct link between the EMT and the gain of epithelial stem cell properties.

Mechanisms of Malignant Progression.
Carcinogenesis. 2008 May 2. [Epub ahead of print]
Weinberg, R.A.*

The Mouse X Chromosome Is Enriched for Multicopy Testis Genes Showing Postmeiotic Expression.
Nat Genet. 2008 Jun;40(6):794-9. Epub 2008 May 4.
Mueller, J.L.*, Mahadevaiah, S.K., Park, P.J., Warburton, P.E., Page, D.C.*, and Turner, J.M.
According to the prevailing view, mammalian X chromosomes are enriched in spermatogenesis genes expressed before meiosis and deficient in spermatogenesis genes expressed after meiosis. The paucity of postmeiotic genes on the X chromosome has been interpreted as a consequence of meiotic sex chromosome inactivation (MSCI)-the complete silencing of genes on the XY bivalent at meiotic prophase. Recent studies have concluded that MSCI-initiated silencing persists beyond meiosis and that most genes on the X chromosome remain repressed in round spermatids. Here, we report that 33 multicopy gene families, representing approximately 273 mouse X-linked genes, are expressed in the testis and that this expression is predominantly in postmeiotic cells. RNA FISH and microarray analysis show that the maintenance of X chromosome postmeiotic repression is incomplete. Furthermore, X-linked multicopy genes exhibit a similar degree of expression as autosomal genes. Thus, not only is the mouse X chromosome enriched for spermatogenesis genes functioning before meiosis, but in addition, approximately 18% of mouse X-linked genes are expressed in postmeiotic cells.

A Yeast Tdp-43 Proteinopathy Model: Exploring the Molecular Determinants of Tdr-43 Aggregation and Cellular Toxicity.
Proc Natl Acad Sci U S A. 2008 Apr 29;105(17):6439-44. Epub 2008 Apr 23.
Johnson, B.S., McCaffery, J.M., Lindquist, S.*, and Gitler, A.D.
Protein misfolding is intimately associated with devastating human neurodegenerative diseases, including Alzheimer's, Huntington's, and Parkinson's. Although disparate in their pathophysiology, many of these disorders share a common theme, manifested in the accumulation of insoluble protein aggregates in the brain. Recently, the major disease protein found in the pathological inclusions of two of these diseases, amyotrophic lateral sclerosis (ALS) and frontal temporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U), was identified as the 43-kDa TAR-DNA-binding protein (TDP-43), providing a molecular link between them. TDP-43 is a ubiquitously expressed nuclear protein that undergoes a pathological conversion to an aggregated cytoplasmic localization in affected regions of the nervous system. Whether TDP-43 itself can convey toxicity and whether its abnormal aggregation is a cause or consequence of pathogenesis remain unknown. We report a yeast model to define mechanisms governing TDP-43 subcellular localization and aggregation. Remarkably, this simple model recapitulates several salient features of human TDP-43 proteinopathies, including conversion from nuclear localization to cytoplasmic aggregation. We establish a connection between this aggregation and toxicity. The pathological features of TDP-43 are distinct from those of yeast models of other protein-misfolding diseases, such as polyglutamine. This suggests that the yeast model reveals specific aspects of the underlying biology of the disease protein rather than general cellular stresses associated with accumulating misfolded proteins. This work provides a mechanistic framework for investigating the toxicity of TDP-43 aggregation relevant to human disease and establishes a manipulable, high-throughput model for discovering potential therapeutic strategies.

An Embryonic Stem Cell-Like Gene Expression Signature in Poorly Differentiated Aggressive Human Tumors.
Nat Genet. 2008. May;40(5):499-507.
Ben-Porath, I.*, Thomson, M.W., Carey, V.J., Ge, R*., Bell, G.W.*, Regev, A., and Weinberg, R.A.*
Cancer cells possess traits reminiscent of those ascribed to normal stem cells. It is unclear, however, whether these phenotypic similarities reflect the activity of common molecular pathways. Here, we analyze the enrichment patterns of gene sets associated with embryonic stem (ES) cell identity in the expression profiles of various human tumor types. We find that histologically poorly differentiated tumors show preferential overexpression of genes normally enriched in ES cells, combined with preferential repression of Polycomb-regulated genes. Moreover, activation targets of Nanog, Oct4, Sox2 and c-Myc are more frequently overexpressed in poorly differentiated tumors than in well-differentiated tumors. In breast cancers, this ES-like signature is associated with high-grade estrogen receptor (ER)-negative tumors, often of the basal-like subtype, and with poor clinical outcome. The ES signature is also present in poorly differentiated glioblastomas and bladder carcinomas. We identify a subset of ES cell-associated transcription regulators that are highly expressed in poorly differentiated tumors. Our results reveal a previously unknown link between genes associated with ES cell identity and the histopathological traits of tumors and support the possibility that these genes contribute to stem cell-like phenotypes shown by many tumors.

*Whitehead Institute for Biomedical Research

Last updated June 3, 2008.