Cynthia A. Sparks

Targeting mTOR: prospects for mTOR complex 2 inhibitors in cancer therapy

Small molecule inhibitors that selectively target cancer cells and not normal cells would be valuable anti-cancer therapeutics. The mammalian target of rapamycin complex 2 (mTORC2) is emerging as a promising candidate target for such an inhibitor. Recent studies in cancer biology indicate that mTORC2 activity is essential for the transformation and vitality of a number of cancer cell types, but in many normal cells, mTORC2 activity is less essential. These studies are intensifying interest in developing inhibitors that specifically target mTORC2. However, there are many open questions regarding the function and regulation of mTORC2 and its function in both normal and cancer cells. Here, we summarize exciting new research into the biology of mTORC2 signaling and highlight the current state and future prospects for mTOR-targeted therapy.

mTOR-Dependent Cell Survival Mechanisms

The mechanistic target of rapamycin (mTOR) kinase is a conserved regulator of cell growth, proliferation, and survival. In cells, mTOR is the catalytic subunit of two complexes called mTORC1 and mTORC2, which have distinct upstream regulatory signals and downstream substrates. mTORC1 directly senses cellular nutrient availability while indirectly sensing circulating nutrients through growth factor signaling pathways. Cellular stresses that restrict growth also impinge on mTORC1 activity. mTORC2 is less well understood and appears only to sense growth factors. As an integrator of diverse growth regulatory signals, mTOR evolved to be a central signaling hub for controlling cellular metabolism and energy homoeostasis, and defects in mTOR signaling are important in the pathologies of cancer, diabetes, and aging. Here we discuss mechanisms by which each mTOR complex might regulate cell survival in response to metabolic and other stresses.

Sarcomas induced in discrete subsets of prospectively isolated skeletal muscle cells

Soft-tissue sarcomas are heterogeneous cancers that can present with tissue-specific differentiation markers. To examine the cellular basis for this histopathological variation and to identify sarcoma-relevant molecular pathways, we generated a chimeric mouse model in which sarcoma-associated genetic lesions can be introduced into discrete, muscle-resident myogenic and mesenchymal cell lineages. Expression of Kirsten rat sarcoma viral oncogene [Kras(G12V)] and disruption of cyclin-dependent kinase inhibitor 2A (CDKN2A; p16p19) in prospectively isolated satellite cells gave rise to pleomorphic rhabdomyosarcomas (MyoD-, Myogenin- and Desmin-positive), whereas introduction of the same oncogenetic hits in nonmyogenic progenitors induced pleomorphic sarcomas lacking myogenic features. Transcriptional profiling demonstrated that myogenic and nonmyogenic Kras; p16p19null sarcomas recapitulate gene-expression signatures of human rhabdomyosarcomas and identified a cluster of genes that is concordantly up-regulated in both mouse and human sarcomas. This cluster includes genes associated with Ras and mechanistic target of rapamycin (mTOR) signaling, a finding consistent with activation of the Ras and mTOR pathways both in Kras; p16p19null sarcomas and in 26–50% of human rhabdomyosarcomas surveyed. Moreover, chemical inhibition of Ras or mTOR signaling arrested the growth of mouse Kras; p16p19null sarcomas and of human rhabdomyosarcoma cells in vitro and in vivo. Taken together, these data demonstrate the critical importance of lineage commitment within the tumor cell-of-origin in determining sarcoma histotype and introduce an experimental platform for rapid dissection of sarcoma-relevant cellular and molecular events.

Product of the oncogene-activating gene Tpr is a phosphorylated protein of the nuclear pore complex

We have identified a component of the human nuclear pore complex and have shown that it is the product of a gene involved in oncogenic activation. A monoclonal antibody raised against purified nuclear matrix proteins recognizes a single protein with an electrophoretic mobility of approximately 300 kDa and stains the nuclear envelope in a punctate pattern typical of nuclear pores. The antibody was used to screen λgt11 human cDNA libraries, and the resulting clones were sequenced and compared to sequences in the Genbank database. An exact match was found with the human tpr (for translocated promoter region) gene, a gene shown previously to be involved in the oncogenic activation of several protein kinases. Double‐label immunofluorescent microscopy with the anti‐Tpr antibody and an antibody to the previously characterized nuclear pore complex protein nup153 confirms that Tpr is localized to the nuclear pore complex. Tpr is located on the cytoplasmic face of the nucleus, as demonstrated by immunofluorescent staining of cells permeabilized with digitonin. Tpr is a 2,349‐amino acid protein with extensive coiled‐coil domains and an acidic globular C‐terminus. The protein contains 10 leucine zipper motifs and numerous sites for phosphorylation by a variety of protein kinases. Immunoprecipitation of Tpr from 32P‐orthophosphate‐labeled cells shows that it is a phosphoprotein. Potential functions for Tpr and possible mechanisms for the transforming activity of Tpr fusion proteins are discussed.