Julian Downward

Intratumor heterogeneity and branched evolution revealed by multiregion sequencing.

BACKGROUND Intratumor heterogeneity may foster tumor evolution and adaptation and hinder personalized-medicine strategies that depend on results from single tumor-biopsy samples. METHODS To examine intratumor heterogeneity, we performed exome sequencing, chromosome aberration analysis, and ploidy profiling on multiple spatially separated samples obtained from primary renal carcinomas and associated metastatic sites. We characterized the consequences of intratumor heterogeneity using immunohistochemical analysis, mutation functional analysis, and profiling of messenger RNA expression. RESULTS Phylogenetic reconstruction revealed branched evolutionary tumor growth, with 63 to 69% of all somatic mutations not detectable across every tumor region. Intratumor heterogeneity was observed for a mutation within an autoinhibitory domain of the mammalian target of rapamycin (mTOR) kinase, correlating with S6 and 4EBP phosphorylation in vivo and constitutive activation of mTOR kinase activity in vitro. Mutational intratumor heterogeneity was seen for multiple tumor-suppressor genes converging on loss of function; SETD2, PTEN, and KDM5C underwent multiple distinct and spatially separated inactivating mutations within a single tumor, suggesting convergent phenotypic evolution. Gene-expression signatures of good and poor prognosis were detected in different regions of the same tumor. Allelic composition and ploidy profiling analysis revealed extensive intratumor heterogeneity, with 26 of 30 tumor samples from four tumors harboring divergent allelic-imbalance profiles and with ploidy heterogeneity in two of four tumors. CONCLUSIONS Intratumor heterogeneity can lead to underestimation of the tumor genomics landscape portrayed from single tumor-biopsy samples and may present major challenges to personalized-medicine and biomarker development. Intratumor heterogeneity, associated with heterogeneous protein function, may foster tumor adaptation and therapeutic failure through Darwinian selection. (Funded by the Medical Research Council and others.).

Targeting RAS signalling pathways in cancer therapy

The RAS proteins control signalling pathways that are key regulators of several aspects of normal cell growth and malignant transformation. They are aberrant in most human tumours due to activating mutations in the RAS genes themselves or to alterations in upstream or downstream signalling components. Rational therapies that target the RAS pathways might inhibit tumour growth, survival and spread. Several of these new therapeutic agents are showing promise in the clinic and many more are being developed.

Mechanisms and consequences of activation of protein kinase B/Akt.

Protein kinase B (PKB)/Akt is a growth-factor-regulated serine/threonine kinase which contains a pleckstrin homology domain. Binding of phosphoinositide 3-OH kinase products to the pleckstrin homology domain results in translocation of PKB/Akt to the plasma membrane where it is activated by phosphorylation by upstream kinases including the phosphoinoside-dependent kinase 1 (PDK1). Activated PKB/Akt provides a survival signal that protects cells from apoptosis induced by various stresses, and also mediates a number of metabolic effects of insulin.

Matrix adhesion and Ras transformation both activate a phosphoinositide 3-OH kinase and protein kinase B/Akt cellular survival pathway

Upon detachment from the extracellular matrix, epithelial cells enter into programmed cell death, a phenomenon known as anoikis, ensuring that they are unable to survive in an inappropriate location. Activated ras oncogenes protect cells from this form of apoptosis. The nature of the survival signals activated by integrin engagement and usurped by oncogenic Ras are unknown: here we show that in both cases phosphoinositide 3‐OH kinase (PI 3‐kinase), but not Raf, mediates this protection, acting through protein kinase B/Akt (PKB/Akt). Constitutively activated PI 3‐kinase or PKB/Akt block anoikis, while inhibition of PI 3‐kinase abrogates protection by Ras, but not PKB/Akt. Inhibition of either PI 3‐kinase or PKB/Akt induces apoptosis in adherent epithelial cells. Attachment of cells to matrix leads to rapid elevation of the levels of PI 3‐kinase lipid products and PKB/Akt activity, both of which remain high in Ras‐transformed cells even in suspension. PI 3‐kinase acting through PKB/Akt is therefore implicated as a key mediator of the aberrant survival of Ras‐transformed epithelial cells in the absence of attachment, and mediates matrix‐induced survival of normal epithelial cells.

Role of Phosphoinositide 3-OH Kinase in Cell Transformation and Control of the Actin Cytoskeleton by Ras

The pathways by which mammalian Ras proteins induce cortical actin rearrangement and cause cellular transformation are investigated using partial loss of function mutants of Ras and activated and inhibitory forms of various postulated target enzymes for Ras. Efficient transformation by Ras requires activation of other direct effectors in addition to the MAP kinase kinase kinase Raf and is inhibited by inactivation of the PI 3-kinase pathway. Actin rearrangement correlates with the ability of Ras mutants to activate PI 3-kinase. Inhibition of PI 3-kinase activity blocks Ras induction of membrane ruffling, while activated PI 3-kinase is sufficient to induce membrane ruffling, acting through Rac. The ability of activated Ras to stimulate PI 3-kinase in addition to Raf is therefore important in Ras transformation of mammalian cells and essential in Ras-induced cytoskeletal reorganization.

PKB/Akt: connecting phosphoinositide 3-kinase to cell survival and beyond

PKB/Akt is a serine/threonine kinase that contains a pleckstrin-homology (PH) domain and is activated in response to growth-factor treatment of cells by a mechanism involving phosphoinositide 3-OH kinase. PKB/Akt provides a survival signal that protects cells from apoptosis induced by various stresses, perhaps explaining its discovery as a retroviral oncogene and its amplification in many human tumours.

Ras and TGFβ cooperatively regulate epithelial cell plasticity and metastasis: dissection of Ras signaling pathways

Multistep carcinogenesis involves more than six discrete events also important in normal development and cell behavior. Of these, local invasion and metastasis cause most cancer deaths but are the least well understood molecularly. We employed a combined in vitro/in vivo carcinogenesis model, that is, polarized Ha-Ras–transformed mammary epithelial cells (EpRas), to dissect the role of Ras downstream signaling pathways in epithelial cell plasticity, tumorigenesis, and metastasis. Ha-Ras cooperates with transforming growth factor β (TGFβ) to cause epithelial mesenchymal transition (EMT) characterized by spindle-like cell morphology, loss of epithelial markers, and induction of mesenchymal markers. EMT requires continuous TGFβ receptor (TGFβ-R) and oncogenic Ras signaling and is stabilized by autocrine TGFβ production. In contrast, fibroblast growth factors, hepatocyte growth factor/scatter factor, or TGFβ alone induce scattering, a spindle-like cell phenotype fully reversible after factor withdrawal, which does not involve sustained marker changes. Using specific inhibitors and effector-specific Ras mutants, we show that a hyperactive Raf/mitogen-activated protein kinase (MAPK) is required for EMT, whereas activation of phosphatidylinositol 3-kinase (PI3K) causes scattering and protects from TGFβ-induced apoptosis. Hyperactivation of the PI3K pathway or the Raf/MAPK pathway are sufficient for tumorigenesis, whereas EMT in vivo and metastasis required a hyperactive Raf/MAPK pathway. Thus, EMT seems to be a close in vitro correlate of metastasis, both requiring synergism between TGFβ-R and Raf/MAPK signaling.

Expression cloning and characterization of the TGF-β type III receptor

The rat TGF-beta type III receptor cDNA has been cloned by overexpression in COS cells. The encoded receptor is an 853 amino acid protein with a large N-terminal extracellular domain containing at least one site for glycosaminoglycan addition, a single hydrophobic transmembrane domain, and a 41 amino acid cytoplasmic tail with no obvious signaling motif. Introduction of the cDNA into COS cells and L6 myoblasts induces expression of a heterogenously glycosylated 280-330 kd protein characteristic of the type III receptor that binds TGF-beta 1 specifically. In L6 myoblasts lacking the endogenous type III receptor, expression of the recombinant receptor leads to an increase in the amount of ligand bound and cross-linked to surface type II TGF-beta receptors. This indicates that the type III receptor may regulate the ligand-binding ability or surface expression of the type II receptor.

Akt Phosphorylates the Yes-Associated Protein, YAP, to Induce Interaction with 14-3-3 and Attenuation of p73-Mediated Apoptosis

We have used an affinity purification method to identify substrates of protein kinase B/Akt. One protein that associates with 14-3-3 in an Akt-dependent manner is shown here to be the Yes-associated protein (YAP), which is phosphorylated by Akt at serine 127, leading to binding to 14-3-3. Akt promotes YAP localization to the cytoplasm, resulting in loss from the nucleus where it functions as a coactivator of transcription factors including p73. p73-mediated induction of Bax expression following DNA damage requires YAP function and is attenuated by Akt phosphorylation of YAP. YAP overexpression increases, while YAP depletion decreases, p73-mediated apoptosis following DNA damage, in an Akt inhibitable manner. Akt phosphorylation of YAP may thus suppress the induction of the proapoptotic gene expression response following cellular damage.

Ras signalling and apoptosis.

Activated Ras proteins have either positive or negative effects on the regulation of apoptosis depending on cell type and other factors. In part, this is due to the ability of Ras to control directly multiple effector pathways, including PI3-kinase, which provides a universal survival signal, and Raf, which can inhibit survival. The mechanisms remain partly unclear, however, especially with regard to Raf effects on apoptosis regulation. Recently Ras has been shown to be able to protect cells from apoptosis either through activation of PKB/Akt via PI3-kinase, or through activation of NF-kappa B.