1.Supplementary Sidebars Supplementary Sidebar 1.1 Supplementary Sidebar 1.2 Supplementary Sidebar 1.3 Supplementary Sidebar 1.4 Supplementary Sidebar 1.5 Supplementary Sidebar 2.1 Supplementary Sidebar 2.2 Supplementary Sidebar 3.1 Supplementary Sidebar 3.2 Supplementary Sidebar 3.3 Supplementary Sidebar 3.4 Supplementary Sidebar 3.5 Supplementary Sidebar 4.1 Supplementary Sidebar 4.2 Supplementary Sidebar 4.3 Supplementary Sidebar 4.4 Supplementary Sidebar 5.1 Supplementary Sidebar 5.2 Supplementary Sidebar 5.3 Supplementary Sidebar 6.1 Supplementary Sidebar 6.2 Supplementary Sidebar 6.3 Supplementary Sidebar 6.4 Supplementary Sidebar 6.5 Supplementary Sidebar 7.1 Supplementary Sidebar 7.2 Supplementary Sidebar 7.3 Supplementary Sidebar 7.4 Supplementary Sidebar 7.5 Supplementary Sidebar 7.6 Supplementary Sidebar 7.7 Supplementary Sidebar 8.1 Supplementary Sidebar 8.2 Supplementary Sidebar 8.3 Supplementary Sidebar 8.4 Supplementary Sidebar 8.5 Supplementary Sidebar 8.6 Supplementary Sidebar 9.1 Supplementary Sidebar 9.2 Supplementary Sidebar 9.3 Supplementary Sidebar 9.4 Supplementary Sidebar 9.5 Supplementary Sidebar 10.1 Supplementary Sidebar 11.1 Supplementary Sidebar 11.2 Supplementary Sidebar 11.3 Supplementary Sidebar 11.4 Supplementary Sidebar 11.5 Supplementary Sidebar 11.6 Supplementary Sidebar 11.7 Supplementary Sidebar 12.1 Supplementary Sidebar 12.2 Supplementary Sidebar 12.3 Supplementary Sidebar 12.4 Supplementary Sidebar 12.5 Supplementary Sidebar 12.6 Each female cell can access information only from a single X chromosome Reproductive cloning demonstrates the extraordinary efficiency of the DNA repair apparatus The network of miRNA-controlled genes Knocking down gene expression with shRNAs and siRNAs Gene cloning strategies Commonly used histopathological techniques The complicated conventions for classifying and naming tumors Is SV40 responsible for the mesothelioma plague? Maintenance of KSHV and HPV genomes in episomal and chromosomal form Re-engineering the retrovirus genome for gene therapy Classic Kaposis sarcoma appears to be a familial disease Viruses like RSV have very short lives Endogenous viruses can explain tumor development in the absence of infectious viral spread Boveri and Hansemann independently hypothesized genetic abnormality as the cause of cancer cells malignant behavior Southern and Northern blotting Genes undergo amplification for a variety of reasons Making anti-Src antibodies presented a major challenge The protozoan roots of metazoan signaling Lateral interactions of cell surface receptors Systematic surveys of phosphotyrosine and SH2 interactions The complexities of understanding RTK signaling The rationale for multi-kinase signaling cascades Non-canonical Wnt signaling The Hippo pathway and control of stem cell proliferation Heterozygosity in the human gene pool Which is more likelyLOH or secondary, independent mutations? The polymerase chain reaction makes it possible to genetically map tumor suppressor genes rapidly The MSP reaction makes it possible to gauge methylation status of promoters Ubiquitylation tags cellular proteins for destruction in proteasomes Krebs cycle enzymes and cancer development Homologous recombination allows restructuring of the mouse germ line The origins of embryonic stem cells Plasticity of the cell cycle clock Chromatin immunoprecipitation (ChIP) Some tumors increase Id concentrations by de-ubiquitylating them Specific targeting of cell cycle regulators by E3 ubiquitin ligases The major puzzle surrounding the RB gene: retinoblastomas UV-B radiation, HPV, and cutaneous squamous cell carcinomas The TUNEL assay Dominant-negative functions of mutant p53 alleles: functional interactions between p53 and its p63 and p73 cousins Some mutant p53 alleles cause highly specific tumors Autophagy is critical to post-fertilization development The use of the TRAP assay and adaptations thereof permits the rapid and quantitative assessment of the levels of the catalytic activity of telomerase enzyme in eukaryotic cells Monoclonal antibodies and fluorescence-activated cell sorting (FACS) How does multi-step tumor progression actually take place? Symbiosis between distinct subpopulations within a tumor Comparative genomic hybridization Are rodent carcinogen tests reliable indicators of danger to humans? Does saccharin cause cancer? How does diet affect colon cancer incidence? Hematopoiesis as a model for the organization of many kinds of tissues Stem cell pools may explain the protective effects of pregnancy The conserved-strand mechanism and protection of the stem cell genome Oxidation products in urine provide an estimate of the rate of ongoing damage to the cellular genome How does red meat cause colon cancer? A convergence of bacterial, yeast, and human genetics led to the discovery of hereditary non-polyposis colon cancer genes1 2 3 4 6 7 9 11 12 14 16 17 18 20 21 22 23 24 25 26 28 29 30 31 32 33 34 35 36 38 41 43 44 45 46 47 48 50 52 53 54 55 56 57 59 61 62 63 64 65 67 69 72 75 76 77

2. Supplementary Sidebar 12.7 Supplementary Sidebar 13.1 Supplementary Sidebar 13.2 Supplementary Sidebar 13.3 Supplementary Sidebar 13.4 Supplementary Sidebar 13.5 Supplementary Sidebar 13.6 Supplementary Sidebar 13.7 Supplementary Sidebar 13.8 Supplementary Sidebar 13.9 Supplementary Sidebar 13.10 Supplementary Sidebar 14.1 Supplementary Sidebar 14.2 Supplementary Sidebar 14.3 Supplementary Sidebar 14.4 Supplementary Sidebar 14.5 Supplementary Sidebar 14.6 Supplementary Sidebar 14.7 Supplementary Sidebar 14.8 Supplementary Sidebar 14.9 Supplementary Sidebar 14.10 Supplementary Sidebar 14.11 Supplementary Sidebar 14.12 Supplementary Sidebar 14.13 Supplementary Sidebar 14.14 Supplementary Sidebar 15.1 Supplementary Sidebar 15.2 Supplementary Sidebar 15.3 Supplementary Sidebar 15.4 Supplementary Sidebar 15.5 Supplementary Sidebar 15.6 Supplementary Sidebar 15.7 Supplementary Sidebar 15.8 Supplementary Sidebar 15.9 Supplementary Sidebar 15.10 Supplementary Sidebar 15.11 Supplementary Sidebar 15.12 Supplementary Sidebar 15.13 Supplementary Sidebar 15.14 Supplementary Sidebar 15.15 Supplementary Sidebar 15.16 Supplementary Sidebar 16.1 Supplementary Sidebar 16.2 Supplementary Sidebar 16.3 Supplementary Sidebar 16.4 Supplementary Sidebar 16.5 Supplementary Sidebar 16.6 Supplementary Sidebar 16.7 Supplementary Sidebar 16.8 Supplementary Sidebar 16.9 Supplementary Sidebar 16.10 Supplementary Sidebar 16.11 Homology-directed repair Localization of growth factors is important for proper heterotypic interactions Ongoing heterotypic signaling in carcinomas Certain highly advanced tumors provide exceptions to the generally observed dependence of carcinoma cells on stroma Myofibroblasts predict clinical progression of cancer A technique for separating stromal from epithelial cells Microvessel leakiness dooms many forms of anti-cancer therapy: optimizing anti-angiogenic treatments The temporary nature of vessel regression created by anti-angiogenesis therapy Kaposis sarcoma cells hold the record for the number of documented heterotypic signals they receive Effects of an anti-VEGF-R monoclonal antibody on the growth of a human tumor xenograft Fibroblasts are heterogeneous and can change dynamically in response to signals Visualization of the dynamics of pathfinding fibroblasts followed by squamous cell carcinoma cells Metastasizing cancer cells often take on hitchhikers while traveling through the blood Instruments for detecting circulating tumor cells Hidden micrometastases are revealed through organ transplantation Wolves in sheeps clothing: when carcinoma cells invade the stroma TGF- works in conflicting ways during tumor progression Dynamics of EMT induction: the EMT may be controlled in some cancer cells exclusively by their own genomes An example of an EMT relatively late in embryonic development Relatively rapid metastatic dissemination of advanced primary tumor cells Our cells devote an enormous number of genes to regulating protein degradation Peritoneovenous shunts provide dramatic support for the seed and soil hypothesis Tooth extractions may occasionally become exceedingly painful Tumor stem cells further complicate our understanding of the metastatic process Does Darwinian evolution accommodate metastasis-specific alleles? Rearrangements of chromosomal DNA segments generate a vast array of antigen-binding domains in antibodies and T-cell receptors Virus-infected cells may not always be recognized by the immune system Bizarre tumors reveal how cancer cells can become infectious agents Mice have proven to be far more useful for tumor biologists than chickens An HPV vaccine protects against many cervical carcinomas An unexpected type of anti-p53 reactivity is often found in cancer patients Immune recognition of tumors may be delayed until relatively late in tumor progression Some paraneoplastic syndromes reveal defective tolerance and overly successful immune responses to tumors TSTAs can arise as by-products of chemical and physical carcinogenesis Are melanomas more antigenic than other tumors? Strategies for cloning genes encoding melanoma TATAs Anti-CD47 therapies hold promise in treating lymphomas and other hematopoietic malignancies Cancer cells may thwart extravasation by circulating T cells Herceptin can be modified to potentiate cancer cell killing Bone marrow transplantation and the treatment of hematopoietic malignancies Whole genome sequencing allows a new attack on tumor cells Modern cancer therapies have had only a minor effect on the overall death rate from the disease Prostate cancers usually do not require aggressive interventiona tale of two countries Clinical practice and our understanding of disease pathogenesis have often been poorly aligned, leading to sub-optimal, often tragic outcomes The ability to assign tumors to specific disease subtypes is critical to the success of drug development p53 germ-line polymorphisms and somatic mutations can complicate the induction of apoptosis by drugs Ras function can be inhibited by interfering with the enzymes responsible for the maturation of the Ras protein Chemical synthesis, compound libraries, and high-throughput screening Evolution can generate huge collections of structurally similar proteins Large-scale screen of the inhibitory effects of a drug on various kinases Epidermal growth factor receptor expression levels predict little about a tumors susceptibility to receptor antagonists Akt/PKB function is controlled by multiple upstream signals78 79 80 81 82 83 84 86 88 89 90 92 93 96 98 99 100 101 102 103 104 105 106 107 108 109 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 131 132 134 135 138 140 142 143 3. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 1.1 Each female cell can access information only from a single X chromosome In the simplest depiction of sex chromosome behavior, each male cell relies on the information carried by its single X chromosome, while each female cell is able to consult both of its X chromosomes. Given the large number of genes present on the X chromosome, this disparity would create substantial physiologic differences between male and female cells, since female cells would, in general, express twice as much of each of the products specified by the genes on their X chromosomes. This problem is solved by 2014 Garland Sciencethe mechanism of X-inactivation. Early in embryogenesis, one of the two X chromosomes is randomly inactivated in each of the cells of a female embryo. This inactivation silences almost all of the genes on this chromosome and causes this chromosome to shrink into a small particle termed the Barr body (Figure S1.1). Subsequently, all descendants of that cell will inherit this pattern of chromosomal inactivation and will therefore continue to carry the same inactivated X chromosome. Accordingly, the female advantage of carrying redundant copies of X chromosomeassociated genes is only a partial one.Figure S1.1 X chromosomes as Barr bodies One of the two X chromosomes in each cell of an early female embryo is randomly inactivated and remains inactivated in all lineal descendant cells. It is visible in the interphase nuclei of these cells, where it remains condensed and associated with the nuclear membrane (white arrow). (From B.P. Chadwick et al., Semin. Cell Dev. Biol. 14:359367, 2003.)TBoC2 b1.10/s1.011 4. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 1.2 Reproductive cloning demonstrates the extraordinary efficiency of the DNA repair apparatus The efficiency of the complex apparatus that repairs DNA (see Chapter 12), the resulting suppression of somatic mutations, and the consequent integrity of the genomes of somatic cells have all been illustrated dramatically by the successes of animal cloning in recent years. In the much-celebrated case of the sheep Dolly, the nuclei from cells taken from the breast tissue of her mother were removed and implanted in egg cells (oocytes) whose own nuclei had previously been eliminated. The resulting cells, containing donor nuclei and recipient egg cytoplasm, were then induced to proliferate and to form embryos (Figure S1.2). The fact that an essentially normal sheep (Dolly) was born many months later, having developed from one of these embryos, demonstrated that the genome in one of 2014 Garland Scienceher mothers mammary gland cells carried no mutations that compromised normal embryologic development. The implied intactness of the genome in the breast cells of Dollys mother indicates a high degree of integrity in DNA replication. During embryonic development, the genome of the fertilized egg that was destined to develop into Dollys mother was copied and recopied dozens of times during the cycles of cell growth and division that intervened between the initial formation of the fertilized egg and the formation, many months later, of the mothers breast tissue. This recopying process, involving DNA replication and a repair apparatus that operated to weed out mutant DNA sequences, was highly effective in preserving an intact somatic cell genome that was capable of programming normal organismic development.Dollys mother cells prepared from mammary glandsingle cell prepared cells fuse by electric shockunfertilized sheep egghaploid nucleus removedegg induced to divideearly embryoembryo transferred to womb of second sheepDolly is bornenucleated eggFigure S1.2 Reproductive cloning and genomic integrity In the initially reported reproductive cloning procedure, the nucleus of an unfertilized egg was removed and the nucleus from a somatic cella mammary gland cell from Dollys motherwas introduced into the enucleated egg. The resulting cell was diploid, having received its entire genetic complement from the mammary gland cell originating with Dollys mother. Being diploid, it was genetically equivalent to a fertilized egg. This reconstituted egg cell was induced to divide by chemical means and proceeded to generate an embryo that could then be implanted in the womb of a foster pseudo-pregnant ewea ewe whose physiologicstate was comparable to that of a naturally pregnant ewe but had been induced in this case by hormonal treatment. This implanted embryo could then develop into a newborn, albeit at low efficiency. Successful generation of a healthy newborn revealed that the donor somatic cell from Dollys mothers mammary gland carried the genome of the species in an essentially intact, functional form. This donor mammary gland cell initially arose after many successive cell divisions during the development of Dollys mother from a natural fertilized egg. The ability of this cells genome to generate Dolly indicated that these many successive cell divisions did not affect its integrity.TBoC2 b1.13/s1.022 5. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 1.3 The network of miRNA-controlled genes Research into the functions of microRNAs makes it increasingly apparent that the molecules are key components of myriad regulatory circuits within cells. Indeed, it is plausible that almost all regulatory circuits rely on one if not several miRNAs in order to modulate or fine tune the signals that they are 2014 Garland Scienceprocessing. The image presented here (Figure S1.3) presents the state-of-the-art in miRNA research in 2008 that focused on miRNAs that affect signaling pathways relevant to cancer pathogenesis. Subsequently reported research has generated a far more elaborate network of interactions.been demonstrated by 2008 to play a role in modulating various Figure S1.3 OncomiRs and cancer pathogenesis cancer-related cell-biological phenotypes; this class of microRNAs Characterizations of microRNAs have revealed that they play have been dubbed oncomiRs. The complexity of this microRNAimportant roles in modulating the expression of proteins in a TBoC2 n1.108/s1.03 regulated circuitry is growing progressively. (From Melanoma wide variety of subcellular regulatory circuits, including those that Molecular Map Project; see http://www.mmmp.org/MMMP/public/ are deregulated during the course of cancer development. This biomap/viewBioMapImage.mmmp) map illustrates the involvement of a series of microRNAs that had3 6. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 1.4 Knocking down gene expression with shRNAs and siRNAs The discovery of microRNAs (miRNAs), as described in Section 1.10, led to an understanding of the complex molecular machinery that allows their processing from primary transcripts in the nucleus and their dispatch into the cytoplasm. There they inhibit the functioning of thousands of cellular mRNA species. At the same time, these discoveries enabled the development of novel experimental strategies in which exogenous miRNAs could be introduced into cells through the use of viral vectors or transfection, whereupon these introduced RNAs exploit the cellular miRNA-processing machinery to produce mimics of endogenously synthesized miRNAs; the mimics then proceed to shut down the operations of targeted mRNA species, partly by blocking translation and partly by driving the degradation of these mRNA molecules (Figure S1.4). This ability to shut down the expression of proteins of expressed genes within cells has become an invaluable tool for cancer researchers who wish to demonstrate the essential role that one or another protein plays in a cell-biological process under study. Extending the earlier nomenclature that described the inactivation of genes in the mouse germlinetermed knockout (see Supplementary Sidebar 7.7)the new procedure has come to be called knockdown. For example, knockdown of the expression of a certain 2014 Garland Sciencepro-apoptotic protein may protect a cell from apoptosis, demonstrating the essential role that the protein plays in orchestrating apoptosis in such a cell. Similarly, knockdown of the expression of a growth factor receptor may render a cell unresponsive to that factor, demonstrating the receptors key role in conferring such responsiveness. While highly useful in principle, in practice the application of this technique has proven challenging in the case of certain genes being targeted for knockdown. Thus, a targeted mRNA may not respond significantly for reasons that are unclear, necessitating the screening of multiple vectors, each targeting a distinct nucleotide segment in the 3 untranslated region of an mRNA under study; in some cases, four, five, or even six alternative siRNAs or shRNAs must be tested before an experimenter discovers one that can reduce protein expression by 90% or more. Independent of these difficulties in achieving significant knockdown is the opposite problem: some siRNAs and shRNA have been found to affect far more mRNAs than their intended targetthe phenomenon of off-target effects. Off-target effects often make it impossible to draw rigorous conclusions from the results of these experiments because of the confounding effects on other mRNAs and proteins operating in unrelated signaling pathways.4 7. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberglentiviral or retroviral vectorCYTOPLASMNUCLEUS polIIIintegrated provirusDrosha pre-shRNA 2014 Garland ScienceFigure S1.4 Suppression of RNA function by RNA interference The process of RNA interference (RNAi) operates normally in cells to produce microRNAs (miRNAs) that suppress mRNA function, as described in Section 1.10 and Figure 1.20. This process can be exploited experimentally to suppress targeted mRNAs. Two major strategies can be used. In the first, a retrovirus or lentivirus vector (see Supplementary Sidebar 3.3) is constructed that uses an RNA polymerase III promoter to drive expression of a transcript that serves as precursor of an shRNA (small hairpin RNA). The resulting preshRNA is processed by the Dicer enzyme into an siRNA (small interfering RNA) of 1921 nucleotides, which is reduced to a single-stranded RNA (ssRNA). The ssRNA associates with the Argonaut protein (Ago2) and other proteins to form a RISC (RNA-induced silencing complex), which anneals to partially homologous target sequences that are usually located in the 3 untranslated region (3UTR) of certain mRNAs. This results either in the cleavage of the mRNA or inhibition of its translation, blocking in both cases expression of the mRNA and thus the gene encoding this mRNA. Because the retroviral or lentiviral provirus is integrated into the cell chromosome and is transmitted heritably to the progeny of an initially infected cell, the expression of a targeted mRNA can be suppressed stably over many successive cell generations. As an alternative strategy, an siRNA of a particular configuration can be chemically synthesized and transfected into a cell. While this is easier than the procedure described above, the targeted mRNA will be suppressed only transiently, since there is no mechanism to ensure the continued synthesis of the siRNA in the descendants of the initially transfected cell.DicerRISCinhibition of translationmRNA cleavages1.045 8. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 1.5 Gene cloning strategies Gene cloning strategies first became practical in the late 1970s, and since that time a diverse array of strategies has been developed. 2014 Garland ScienceFigure S1.5 illustrates the logic of gene cloning that is common to many of these strategies.fragment genomeintroduce fragments separately into vector arms of vectoramplify each fragment separately in a bacterial colonyidentify and amplify inserted fragment of interestgenome segment (carrying gene of interest) has been clonedFigure S1.5 Molecular cloning of genes Many versions of the gene cloning procedure have been developed since this technology was first invented in the 1970s. Pictured is an outline of one version. The genome from an organism (e.g., humans) is fragmented into relatively small segments, often several tens of kilobases long (top); each DNA segment is linked to the DNA of a vector (gray arms), yielding a recombinant vector. Each recombinant vector is then inserted independently into a bacterium, which is expanded into a colony containing many thousands of bacteria. The bacterial colony bearing the vector with the gene of interest is identified and the vector is retrieved, yielding millions of copies of this genomic fragment (bottom right).TBoC2 b1.21/s1.056 9. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 2.1 Commonly used histopathological techniques Several techniques are used frequently to mark structures in the fields viewed through a light microscope in order to resolve tissue architecture, the subcellular structuresFigure S2.1 Some frequently used techniques for staining histopathological sections While certain modern microscopic techniques allow visualization of tissues at various depths of focus (sometimes termed the Z axis), the great majority are limited to viewing at a single depth, either in cell monolayers or in tissue sections. The latter are prepared by sectioning tissues that have been stabilized by certain fixatives, such as formaldehyde, glutaraldehyde, or ethanol, which permeabilize cells and cross-link molecules in the biological sample prior to its being embedded in paraffin wax and sectioned; this embedding increases the mechanical strength of the sample, thereby facilitating sectioning. Alternatively, a tissue sample may be frozen and sectioned. In both cases, the resulting tissue sections are then deposited on glass slides. The magnification of the images (e.g., 20) is then represented as the size of an object in an image of a microscopic field divided by its actual size; this factor may then be further increased or decreased as the initially viewed image is printed or presented in digital form. (A) Hematoxylin and eosin (H&E) staining reveals complex tissue architecture in this section of a human basal-like carcinoma of the breast (40). Hematoxylin stains nuclear structures, notably the histones in the chromatin, blue; in contrast, eosin stains cytoplasm, connective tissue, and extracellular structures, such as extracellular matrix, pink. Note the two major types of tissue organization here: the carcinoma cells have large dark purple nuclei and are assembled in continous aggregates while the nonneoplastic cells forming the loose, fibrous stroma have small very dark nuclei. (B) Use of Massons trichrome stain allows a variety of structures to be visualized with a single staining procedure. In this micrograph (20) of experimentally transformed human mammary epithelial cells growing as a tumor xenograft in a mouse, erythrocytes (red blood cells) appear bright red, collagen appears blue, cytoplasms appear light pink, and cell nuclei appear dark pink (C) Immunohistochemistry (IHC) depends on the ability to conjugate (link) an antigen-specific antibody with an enzyme, usually horseradish peroxidase; this enzyme uses added hydrogen peroxide (H2O2) to oxidize an added chemical substrate, usually generating a dark brown or black product. In this image (40), the section of a human basal-like breast tumor has been immunostained using an antibody that binds the p53 tumor suppressor protein, which, in mutant form, accumulates in high concentrations in cell nuclei, revealing them here as dark ovals. The section was then stained with hematoxylin which was used here as a counterstain to reveal remaining structures in the tumor, seen here as light blue. Nuclei stained with hematoxylin but not with the anti-p53 peroxidase-conjugated antibody are mostly normal, non-neoplastic cells of the tumor-associated stroma. (D) Normal human mammary gland tissue has been 2014 Garland Sciencewithin individual cells, proteins that form these various structures, and even specific genes borne by chromosomal DNA (Figure S2.1).stained here using the technique of immunofluorescence (IF), in which antigen-specific antibodies have been conjugated to fluorescent dyes (sometimes termed fluorophores) that emit light at various wavelengths after being excited with light of different, specific wavelengths. This allows the staining with multiple distinct antigen-specific antibodies (in this case three) that enables the concomitant visualization of multiple distinct antigens and thus the cells that express them. In this micrograph (63), cells expressing the CD44 antigen have been stained with a conjugated antibody that generates a pink/purple signal, cells expressing the CD24 antigen have been stained with an antibody that generates a blue signal, while cells expressing -smooth muscle actin have been stained with an antibody that generates a green signal. The blue mammary epithelial cells are arrayed as the lining of ducts that have a dark lumen (cavity) in their center. (E) An alternative combination of antibodies allows this visualization of human basal-like breast cancer at 40 magnification. In this case, the anti-p53 antibody has been conjugated to a fluorescent dye emitting a blue signal, an antibody recognizing the BRCA1 protein has been conjugated with a dye emitting a red signal (yielding small red dots in cell nuclei), while an antibody recognizing the PTEN tumor suppressor protein has been stained with an antibody that emits green light. This image indicates that the neoplastic cells with pinkish blue nuclei generally have lower levels of the BRCA1 protein than do the non-neoplastic cells of the tumor stroma, which lack mutant p53 protein and thus the pinkish blue nuclei. (F) In this micrograph (63), immunofluorescence staining has been combined with fluorescence in situ hybridization (FISH), which allows the detection of DNA sequences in the section by annealing (hybridizing) sequence-specific DNA probes that have been coupled with specific fluorescent dyes; the combined staining is sometimes termed immunoFISH. Here, an anti-p53 antibody stains carcinoma cell nuclei dark blue, while specific DNA probes have detected DNA sequences associated with the centromeres of Chromosome 10 (CEP10, red) and Chromosome 17 (CEP17, green), as well as the genes encoding the PTEN (yellow) and BRCA1 (light blue) proteins. (G) Monkey kidney cells growing in monolayer culture were forced to express a protein that caused them to undergo cellcell fusion, generating a multinucleated cell, sometimes termed a polykaryon or syncytium (center). The nuclei were stained with DAPI (4,6-diamidino-2phenylindole), a dye that emits blue fluorescence when it binds to dsDNA (double-stranded DNA), while the actin cytoskeleton was stained with phalloidin, which binds actin filaments and in this case has been linked to the green fluorescent dye FITC (fluorescein isothiocyanate). (A,CF, courtesy of F. Martins and K. Polyak. B, courtesy of S. McAllister. G, courtesy of S. Rozenblatt.)7 10. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg (A)(B)(C)(D)(F) 2014 Garland Science(E)(G)8 11. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 2.2 The complicated conventions for classifying and naming tumors There are several hundred distinct cell types forming the specialized tissues in the human body. Human tumors that arise in the cells of these various tissues exhibit a wide spectrum of phenotypes that defy categorization by any single classification scheme that utilizes either purely morphological or purely molecular criteria. Consequently, human tumors are currently classified by a combination of their histological, clinical, and molecular features. A purely molecular taxonomy may well emerge in the future, but for the moment we are forced to deal with the complex classification schemes that have developed over the past century. The rationale behind the currently used approaches is outlined below. The great majority of human tumors arise from epithelial tissues. While the term cancer is generally used to refer to all malignant human tumors, the term carcinoma specifically refers to human tumors that arise from epithelia. The term adenocarcinoma is a further subdivision of epithelial cancers that arise from single-cell-layer or two-cell-layer epithelia that share key features in their form and function. For example, these epithelia form glands in which the epithelial cells exhibit an apical basal polarity, with the apical end oriented toward the lumen (the central space enclosed by the gland) and the basal end oriented away from the lumen. In general, these cells secrete substances into the lumen and some may also absorb substances from it. This explains why the prefix adeno- (which is derived from the Greek word for gland) is used to distinguish the above tumors from other carcinomas that arise in epitheliasuch as those of the skin and bladderthat act as a protective barrier rather than facilitator of molecular transport. Benign epithelial tumors are in general referred to as adenomas. It is notable that the majority of human cancers are adenocarcinomas arising from lung, breast, prostate, pancreas, liver, colon, uterus, ovary, and so forth. Precisely why adenocarcinomas account for such a disproportionate share of human tumors is not easily explained by invoking either the proportion of the human body that these epithelia represent or the proliferation rate of their constituent cells. Nonepithelial tumors are dramatically different from epithelial tumors in several important aspects. Thus, when referring to 2014 Garland Sciencethese other tumors, a uniform terminology, comparable to the adenoma and adenocarcinoma terminology used for epithelial tumors, is often replaced by a differentiation state-based system, that is, one that is based on the degree of differentiation and tumor progression. For example, benign nonepithelial tumors that arise from the mesenchymal cells of the stroma are designated by adding the suffix -oma to the name of the normal differentiated tissue that they resemble; hence, lipoma is the term for fat-cell tumors. The suffix -sarcoma is used for the corresponding malignant tumorsliposarcoma, in this example that appear to arise in the same tissue. The hematopoietic neoplasms are classified based upon their physical appearance, specifically, lymphomas (solid masses) or leukemias (individual cells suspended in the blood), regardless of their benign or malignant behavior. Much of this terminology developed independently for each tissue type and reflects the history of pioneering research for each neoplasm. By now, the terms in common use are so embedded in clinical oncology that they are essentially impossible to change. Regardless of these quirks, these broad categories can teach important lessons about the nature of tumors that arise in different tissue types. For example, there is a great difference between benign epithelial and stromal tumors, both of which may be called -omas. Epithelial adenomas are often intermediaries in a multi-step progression that begins with fully normal cells and ends with malignant adenocarcinomas, as is discussed in Chapter 11. In contrast, certain benign stromal -omas may persist stably in a benign state without further progression and may thus behave very differently from malignant sarcomas arising in the same tissues. In addition, there is a great difference in the types of genetic lesions that drive malignancy in these two broad classes. Most nonepithelial tumors are typically associated with a single, dominant genetic event, such as a specific gene translocation. Often, these translocations are so uniform (recurrent) among different patients and so distinctive that their presence is determined in order to help make a definitive diagnosis of the specific type of tumor being examined. Examples of these tumors are listed in the accompanying Table S2.1 below. In contrast, it is generally very difficult to find any single mutation or translocation that is uniformly present in most cases of a given epithelial cancer type, with the K-ras mutationsTable S2.1 Examples of malignant nonepithelial tumors and their characteristic genetic lesions TumorTranslocationInvolved genesCMLt(9;22)(q34;q11)Bcr-AblEwings sarcomat(11;22)(q24;q12)EWS-FLI1Clear-cell sarcomat(11;22)(p13;q12)EWS-WT1Myxoid chondrosarcomat(9;22)(q22;q11-12)EWS-ATF1Alveolar rhabdomyosarcomat(2;13)(q35;q14) t(1;13)(q35;q14)PAX3-FKHR PAX7-FKHRSynovial sarcomat(X;18)(p11.2;q11.2)SYT-SSX1; SSX2Myxoid liposarcomat(12;16)(q13;p11)CHOP-TLS(FUS)Congenital fibrosarcomat(12;15)(q13;q25)ETV6-NTRK3Text and table courtesy of Tan A. Ince.9 12. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg in pancreatic carcinomas and HER2/neu amplifications in a subset of breast cancers being rare exceptions. Epithelial malignancies are typically driven by multiple, accumulated genetic events that are present in different combinations in different tumors and follow the well known multi-hit model of stepwise progression to cancer (see Chapter 11). Tumors that arise in the nervous system represent yet another special category. The cells of the brain and peripheral nerves are derived from the specialized neural ectoderm of the early embryo (see Figure 2.5) and thus differ from non-neural epithelia and mesodermal derivatives (that is, stromal and hematopoietic cells). Due to their distinct embryologic origin, the neuroectodermal tumors that arise in the central and 2014 Garland Scienceperipheral nervous system are considered to be a fourth category of tumors. Nature knows no fixed rules, however, and there are some tumors that are difficult to classify from this simple perspective. For example, choroid plexus cells that line the ventricles of the brain are also derived from the neural ectoderm, but they give rise to tumors with an epithelial phenotype. Finally, some tumors can develop into subpopulations of neoplastic cells exhibiting distinct cell phenotypes, yielding admixtures of epithelial and even stromal cell types. The prototypical example of this class of tumors is provided by teratomas, which are composed of tissues from multiple germ cell layers and hence are suspected to arise from germ or stem cells.10 13. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 3.1 Is SV40 responsible for the mesothelioma plague? The mesothelium is the membranous outer lining of many internal organs and derives directly from the embryonic mesoderm. Mesotheliomastumors of various mesothelial surfaces, notably the pleural membranes of the chestwere virtually unknown in the United States in the first half of the twentieth century. Beginning in about 1960, however, their incidence began to climb steeply. By the end of the century, the annual incidence in the United States approached 2500 and was attributed mostly to asbestos exposure, specifically a subtype termed crocidolite. The disease is seen frequently among those who have worked with asbestos, which was used as a heatresistant insulating material until being banned in the 1980s. However, 20% of mesothelioma patients have no documented exposure to asbestos, a fact that has provoked a search for other etiologic (causative) agents. SV40 is a plausible etiologic agent of mesothelioma. By 2003, forty-one laboratories across the world had reported SV40 DNA, RNA, or protein in mesothelioma cells. Traces of the virus are otherwise rarely found in human tumors, with the exception of certain types of brain tumors. Cultured human mesothelial cells are readily infected by SV40, and this infection leads rapidly to their immortalization, that is, to the ability of these cells, which usually have limited proliferative potential in culture, to multiply indefinitely. 2014 Garland ScienceThe presence of SV40 contamination in poliovirus vaccine stocks has raised concerns that mesothelioma may have been induced in many individuals as an unintended side effect of poliovirus vaccination. However, there are many individuals with mesotheliomas who are highly unlikely to have been exposed to these vaccines. Moreover, the viral T-antigen protein, whose presence is invariably observed in SV40-transformed cells (see Section 3.6), is rarely detected in mesothelioma tumors. Also, attempts at demonstrating viral DNA have usually yielded DNA segments that are indicative of laboratory artifacts, for example, contamination of mesothelioma tumor samples by laboratory stocks of SV40 or by recombinant DNA plasmids engineered to include segments of the viral genome. The discoveries of such contaminations provide increasing ammunition for those who are skeptical of SV40s role in mesothelioma pathogenesis. As many as 85% of humans are known to be infected with two viruses that are closely related to SV40JC and BKand antisera that recognize the capsids of these viruses cross-react with the capsid of SV40, explaining many of the claims that SV40 is often present in human tissues. In immunosuppressed individuals, notably AIDS patients, JC virus can cause a fatal brain degenerationprogressive multifocal leukoencephalopathy (PML). And in patients who are immunosuppressed because they are organ transplant recipients, BK virus has been implicated in the causation of certain carcinomas (see, for example, Figure 3.14B).11 14. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 3.2 Maintenance of KSHV and HPV genomes in episomal and chromosomal form Both herpesvirus and papillomavirus genomes are able to persist for years in a virus-infected tissue in an episomal form, that is, as molecules that are not integrated into the host-cell chromosomal DNA. In the case of Kaposis sarcoma herpesvirus (KSHV), this is achieved through the actions of its LANA protein (Figure S3.1A), which binds to terminally repeated sequences in the viral genome as well as to the histone molecules forming the core nucleosome (see Figure 1.19). This enables maintenance of the viral genome even though direct covalent linkage between viral and host DNAs is absent. A similar dynamic operates in human papillomavirus (HPV)infected tissues. Following initial infection, HPV-infected cells and viral genomes in general are cleared from the infected tissue within a period of weeks or months by the immune system. However, in a minority of infected individuals, the HPVs persist, establishing chronic infections in epithelial tissues and maintaining their genomes, once again, in an episomal state, apparently for years if not decades. These viral infections sometimes trigger low- and then high-grade squamous intraepithelial lesions (LSILs, HSILs) of the cervix; the number of episomal HPV genomes per cell generally increases during this progression and, depending on the subtype of HPV virus, may reach more than several hundred copies per infected cell. On rare occasion, these chronic infections may trigger the onset of frank malignanciescervical carcinomas; squamous cell carcinomas triggered by HPV can also arise in the oropharynx. In the genomes of the cervical carcinoma cells, oncogenic fragments of the HPV genomes are invariably found in an integrated state, while the episomal HPV genomes are generally no 2014 Garland Sciencelonger detectable. This integration is achieved by mechanisms similar to those used by SV40 to establish its genome in cells that it has transformed (see Figure 3.16/n3.103) and involves nonhomologous recombination between viral genomes and hostcell chromosomal sequences. This chromosomal integration, as was the case with SV40, ensures the perpetuation of the HPV genome fragments in descendant cells. The HPV genomes seem to be integrated preferentially in chromosomal common fragile siteschromosomal regions (>100) scattered throughout normal genomes and exhibiting relatively high genetic instability, including susceptibility to dsDNA breakage. This integration has a second functional consequence: the molecular steps that lead to integration usually result in the deletion or inactivation of certain viral genes that are needed for viral replication of episomal viral DNA, and of other genes whose products operate to repress expression of the virus-encoded oncoproteins (Chapters 7 and 8). Without this repression, the viral oncoproteins are expressed by the integrated viral genomes at far higher levels, thus driving the progression of pre-neoplastic virus-infected cells into carcinoma cells. The repression of HPV oncoprotein expression is the task of the viral E2 protein, which has a second, ostensibly related function: it acts to link the unintegrated viral genome to a cellular protein that is bound to chromatin during mitosis (Figure S3.1B). This ensures the tethering of the viral genome to chromosomes during mitosis (Figure S3.1C). When this tethering is blocked experimentally, the viral genomes are unable to hitchhike with the chromsomes during mitosis and consequently are largely left in the cytoplasmic portion of the mitotic cells, which leads to their rapid loss following multiple successive cellular growthand-division cycles.12 15. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg (A) 2014 Garland Science(B) DNAN-terminal domain of E2C-terminal domain of E2H3 Brd4 chromatin proteinH2ACN hingeH4 LANAN123binding to viral DNAH2B (C)E2 functional~175 on chromosomes E2 blockedFigure S3.1 Tethering of episomal viral genomes to host-cell chromatin Certain DNA viruses maintain chronic infections through their ability to maintain their genomes stably but in an unintegrated (episomal) configuration over many successive cell generations. They do so by synthesizing viral proteins that are able to physically link the viral nucleoprotein to the chromatin of the infected host cell when that cell passes through mitosis, ensuring transmission of the viral episomal genomes to both daughter cells. (A) In the case of KSHV/ HHV-8, the agent of Kaposi sarcoma, the virus makes a protein termed LANA (latency-associated nuclear antigen; blue), that is able, on the one hand, to associate with a nucleosome at the interface between its H2A and H2B histones (see Figure 1.19) and, on the other, to bind terminal repeat sequences in the viral DNA genome. (B) Human papilloma virus type 16 (HPV-16) uses the N-terminal domain of its E2 protein (light green) to tether the episomal viral DNA genome to the Brd4 chromatin-remodeling protein (blue), which is bound, in turn, to cellular chromatin during mitosis, enabling the viral genome (tethered to the C-terminal domain of E2) to hitchhike with the cellular chromosomes during mitosis. (C) The association of HPV-16 genomes with metaphase chromosomes can be gauged using HPV-16-specific DNA probes in FISH (fluorescence in situ hybridization) analyses. The chromosomes are stained here in red, while the viral genomes are detected as yellow dots if they are located above the metaphase chromosomes and as green dots if they are not located above the chromosomes. In the upper panel, the E2 protein is functioning normally in chromosomal tethering, whereas in the lower panel, its ability to bind to the Brd4 chromatin-associated protein has been blocked. As indicated, enumeration of the viral genomes above chromosomes reveals a drastic decrease in the metaphase chromosome-associated viral genomes when E2 tethering function is blocked. (A, from A.J. Barbera et al. Science 311:856861, 2006. B and C, upper, from E.A. Abbate, C. Voitenleitner, and M.R. Botchan. Mol. Cell 24:877889, 2006. C, lower, courtesy of C. Voitenleitner and M.R. Botchan.)~5 on chromosomesTBoC2 s3.0113 16. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 3.3 Re-engineering the retrovirus genome for gene therapy The technology of gene therapy has depended on the ability to insert genes into cells or tissues that lack functional versions of those genes, with the intent of reversing the genetic and thus phenotypic defects. The technique of transfection, in which naked DNA is introduced into cells via a variety of techniques (for example, see Figure 4.1) is relatively inefficient and the introduced DNA is integrated into the chromosomal DNA of the recipient cell in a haphazard fashion, resulting in multiple copies of introduced DNA, some of which Figure S3.2 Construction of a retroviral vector While a variety of retroviral-like genomes have been used to construct viral vectors, the basic schemes used in all of these constructions are quite similar. (A) The dsDNA provirus of a retrovirusthe product of reverse transcriptionis introduced into a cloning plasmid and modified subsequently by the standard techniques of recombinant DNA. Note that the provirus contains two identical copies of a segment (red) termed the LTR (long terminal repeat) at its ends products of the reverse transcription process. The presence of these LTRs is critical for the transcription of the provirus by the cellular RNA polymerase II. Restriction enzymes are then used to cleave out viral genes (dashed lines) and to replace them with a gene-of-interest (orange) that will be transduced by the future retroviral vector. In addition, a gene encoding a selectable marker (purple) is introduced. Once expressed in a vector-infected cell, this marker gene can be used to ensure that this cell will be protected from the toxic effects of an applied drug (for example, neomycin), while uninfected cells lacking this marker gene (and thus the remainder of the vectors genome) will be killed off. This strategy can guarantee that the only surviving cells in a large population of cells that have been exposed to this re-engineered virus will be those cells that have been successfully infected by the viral vector. (B) The introduction of two reading frames into this vectorthat encoding the geneof-interestcreates a problem, since in general, the translation of a mammalian mRNA can only be initiated at one site near its 5 end. Hence, when this bicistronic provirus (carrying two genes) is transcribed into an mRNA, ribosomes may only be able to translate the 5-proximal reading frame of this mRNA. This problem can be addressed in at least two ways. Thus, the selectable marker can be introduced in place of the viral env gene; following transcription of a normal retroviral provirus, a certain proportion of the primary pre-mRNA transcripts are routinely processed by splicing in a fashion that causes the viral gag and pol genes to be excised, thereby juxtaposing the env gene (or a gene introduced into its place) with the 5 end of the resulting mRNA. By replacing the viral env gene with an introduced gene-ofinterest, spliced mRNA can then serve as a template for translation by ribosomes, since this gene has now been juxtaposed with the 5 end of the mRNA. A more frequently used strategy, shown here, is to introduce an IRES (internal ribosome entry site; blue) between the two introduced genes just upstream of the (in this case) selectable marker. This IRES segment overrides the normal mechanisms preventing ribosomes from initiating translation at more than one site of an mRNA template; instead, in the presence of an IRES segment, a ribosome can now initiate translation in the middle of an mRNA in addition to its normal 5-proximal site of initiation. In principle, if this provirus is introduced into a cell (for example, via gene transfer/transfection, see Figure 4.2), it can be transcribed by the cellular RNA polymerase II and thus create a polyadenylated RNA molecule that is exported into the cytoplasm. However, this 2014 Garland Scienceare fragmented at random sites during the integration process. Many of these problems are addressed by the use of retrovirus genomes as vectors to transduce (carry) cloned DNA sequences into recipient cells (Figure S3.2). These vectors take advantage of the high efficiency of integration of retroviral DNA and the resulting stable establishment in the recipient cell of integrated proviruses that carry intact copies of the introduced DNA. This technology is limited, however, by the carrying capacity of a retroviral vector, which is only able to accommodate four to five kilobases in introduced sequences in its genome. RNA cannot leave the cell because it cannot be packaged by viral proteins, since some and perhaps all of the viral protein-encoding genes have been deleted during the construction of this viral vector. (The strategy of leaving these genes present and intact in the re-engineered viral genome is not viable, since retroviral virions can only package an RNA molecule of ~10 kb in length. Hence, viral genes must be excised to make room for inserted genes-ofinterest and selectable marker genes.) This problem necessitates co-transfecting the re-engineered vector DNA together with wild-type proviral DNA, which expresses the requisite virion proteins. Such co-transfection can ensure that many of the cells in a population exposed to these two DNAs will take up and express both DNAs. In such co-transfected cells, the viral proteins expressed by the wild-type viral genome can now package the RNA transcribed from the re-engineered vector DNA, resulting in infectious virions that can then be used to infect a variety of target cells, thereby transducing (introducing) the gene-of-interest into these cells. (C) The above strategy is effective in transducing genes-of-interest into cells that previously did not express them. However, it has a major drawback: these initially infected cells may become co-infected with wild-type viral genomes, and the resulting co-infected cells may then release progeny particles that can spread further in a population or in the body of an infected animal during a second infectious cycle. This creates a major problem if the transduced gene-of-interest happens to be a gene that may be toxic or oncogenic, since subsequently occurring, endless rounds of infection might, in principle, spread this gene in an uncontrollable fashion, thereby creating a biohazard. Alternatively, the infectious spread of the wild-type virus may also create a hazard, since nontransforming retroviruses may be tumorigenic if they create large numbers of infected cells and thereby inadvertently activate by insertional mutagenesis infected proto-oncogenes (see Section 3.11). In order to prevent these possibilities, a strategy has been devised that allows the viral vector to be introduced into an initially infected cell but precludes further infectious spread from that cell to yet other cells in second and subsequent rounds of infection. This strategy depends on the creation of packaging cells, which express the three viral mRNAs (encoding the viral gag, pol, and env proteins) from three unlinked viral genes, none of which is part of a provirus. Such cells can package the re-engineered viral vector RNA, because they synthesize all the proteins required for assembly of an intact virion and thus for virion infectivity. However, when another cell is subsequently exposed to the infectious virions leaving this cell, this cell will acquire the re-engineered vector genome and will express its genes, but this cell will be unable to generate a new generation of infectious virions, since no intact viral genomes were ever co-introduced into such cells. Therefore this infected cell, although genetically altered, will not become the source of infectious virus particles that can launch subsequent rounds of infection.14 17. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg (A)gagMLV RNA 5polMLV RNA(B)envAAAAAA...3dsDNA LTRreverse transcriptionLTR introduce IRES segmentdsDNAs LTR 2014 Garland ScienceLTR gene of interestexcise viral genesselectable markerLTR LTRLTR IRESLTR introduce gene of interest+ viral vector with introduced sequencesgene of interest LTRwild-type proviral DNA co-transfect engineered vector with wild-type proviral DNALTRtranscription of transfected proviral DNAintroduce selectable marker gene selectable marker gene LTRpackaging of both RNAs into infectious virionsLTR(C) engineered viral vector DNAmRNAs expressed by packaging cells 5transfect vector DNA into packaging cells that express wild-type viral mRNAs from three unlinked genes5envgag5AAAAAA...3 pol AAAAAA...3 AAAAAA...3infectious virions that can transduce vector into cellssecondary spread from initially infected cells cannot occur because these virions lack the gag, pol and env genesTBoC2 s3.0215 18. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 3.4 Classic Kaposis sarcoma appears to be a familial disease Some virus-induced malignancies are limited largely to small subpopulations and thus resemble familial cancer syndromes. We will encounter many of these syndromes in Chapters 7, 8, 9, and 12. There, we will learn that inheritance of mutant alleles of tumor suppressor genes or DNA repair genes can create a strong, inborn cancer predisposition. Prior to the onset of the AIDS epidemic, the disease of Kaposis sarcoma (KS)a malignancy of cells related to those forming the endothelial lining of lymph ductswas confined largely to small subpopulations, notably men of Mediterranean and Jewish descent. This resembled a familial cancer, in which cancer-predisposing alleles were present only in the gene pools of certain ethnic subpopulations. After the onset of the AIDS epidemic, however, KS became 1000-fold more common, and at least this form of KS could be associated with an infectious agenthuman herpesvirus-8 (HHV-8), also known as KSHV. This virus, along with a number of other infectious agents, is an opportunistic pathogen that thrives in the bodies of those lacking a functional immune system. Because of the AIDS epidemic in Africa, KS has now become the fourth most common infection-induced cancer worldwide. The virology of HHV-8 failed to explain how the classic, pre-AIDS KS is transmitted in immunocompetent populations even though these tumors could also be associated with HHV-8 infections. Moreover, it was unclear why this disease should be confined to small populations rather than spreading, like other viral infections, through large populations. Recent research has revealed that different substrains of HHV-8 (as defined by sequence polymorphisms in viral DNAs) are present in different subpopulations of Jews; one substrain predominates among Ashkenazic Jews (of recent European descent), while a second is common among Sephardic Jews (of North African and Middle Eastern descent). Both populations have infection rates that are 2014 Garland Scienceas much as 10 to 20 times higher than in non-AIDS Western populations. Provocatively, within these subgroups, the transmission of specific HHV-8 substrains is correlated with inheritance of certain mitochondrial DNA polymorphisms far more strongly than with inheritance of Y-chromosome polymorphic markers. Mitochondrial DNA is transmitted maternally, indicating that maternal transmission of virus (occurring possibly via saliva) has played a major role in creating pockets of disease in family lineages that are likely to extend back to founder populations that existed more than 2000 years ago. [Another maternal transmission route may explain the high incidence of adult T-cell leukemia (caused by the HTLV-I retrovirus) in southern Japan (see Section 3.12).] Unexplained by these findings is the fact that KS is confined in the non-AIDS populations to males a mystery that remains unresolved. Hence, certain geographically and ethnically localized malignancies, such as classic KS and adult T-cell leukemia, are actually due to viruses that spread poorly horizontally (that is, from one adult to another) but can be transmitted vertically (between parent and offspring) through long-term, intimate contact. This echoes the behavior of certain strains of mice that have high rates of breast cancer. As first shown in Bar Harbor, Maine, in 1933, transmission of disease from parent to offspring occurred when females of high-incidence strains were mated to males of low-incidence strains, but not following the reverse matings. Also, when female pups of high-incidence strains were transferred to low-incidence foster nursing mothers within 24 hours of birth, only 8% eventually developed breast cancer, compared with a 92% incidence exhibited by mice that had been nursed by mothers from a high-incidence strain. This led to the conclusion that this breast cancer susceptibility was transmitted from one generation to the next by a milk-borne infectious agent, which was later identified as mouse mammary tumor virus (MMTV).16 19. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 3.5 Viruses like RSV have very short lives Circumstantial evidence indicates that infection of a chicken cell by a virus like avian leukosis virus (ALV) resulted in the introduction of a copy of the chicken c-src gene into the ALV genome, creating the hybrid virus that we term RSV. This recombination event seems to have occurred only once in the twentieth century; that is, all src-containing chicken retroviruses appear to be the descendants of the RSV that arose on one occasion in a chicken coop in about 1909. The discovery of RSV depended on a fortuitous circumstance: Peyton Rous was able to study the virus only because a Long Island, New York, chicken farmer brought him a prized, tumor-bearing hen with the hope that Rous would be able to cure the bird of its cancer (see Figure 3.1B). 2014 Garland ScienceThe presence of the acquired src gene in the viral genome confers no obvious growth advantage on this virus, and RSV is not naturally spread from animal to animal. Moreover, similarly configured retroviruses, which also carry both viral and cellular sequences in their genomes, spread poorly from one host animal to another, often because they have deleted essential viral replication genes from their genomes in order to accommodate acquired oncogenes of cellular origin. For these reasons, it seems clear that hybrid retroviruses such as RSV arise through rare genetic recombination events within infected animals, create tumors in these animals, and usually disappear when these animals die, unless an alert chicken farmer or slaughterer happens to discover the tumors and pass them on to an interested virologist.17 20. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 4.1 Endogenous viruses can explain tumor development in the absence of infectious viral spread The exposure of a mouse or chicken to a retrovirus often results in the infection of a wide variety of cell types in the body including, on occasion, the cells in the gonadsovaries or testes. Infections of cells in these organs can result in the integration of a retroviral provirus (see Section 3.7) into the chromosomes of cells that serve as precursors to either sperm or egg. Such proviruses become established in a genetic configuration thatm.m.dom.m.m.mol.transmission of infectious virus particlesis equivalent to that of the cellular genes carried by the sperm or egg. Therefore, when these gametes participate in fertilization, the provirus can be transmitted to a fertilized egg and thus to all of the cells of a resulting embryo and adult (Figure S4.1). This provirus might now become ensconced in the genome of all animals that descend from the initially infected animal. Such a provirus would be termed an endogenous virus to distinguish it from viruses that are transmissible from one individual to the next via infection.Mus musculus outbred subspecies Mus musculus laboratory strains(B)m.m.cas. m.m.mus.(A)a b c d e f g h i j kkb 9.4infection of germ cells, e.g., in testes6.64.4 production of sperm carrying a provirusproduction of a zygote carrying a provirus2.3 2.0development of animal carrying a provirus in all of its cells(C) 5 LTR3 LTRK10 probe transmission via germ line to descendants1 2 3 4 5 6 7 8 9 1012q14 transcriptional activation of an endogenous virus in one cell11q223q24spread of virus via infection of cells throughout the body108b 109 115 2014 Garland ScienceFigure S4.1 Origin of endogenous retroviruses (A) These viral genomes arise when retroviruses (red dots) succeed in establishing a systemic infection in an organism (for example, a mouse) and infect, among other cells, a precursor cell of gametessperm or eggs. Once a resulting provirus (green rectangle) becomes integrated in the genome of a gamete (in this case sperm), it may be introduced into the genome of a fertilized egg and thereafter be distributed to all cells (green dots) of the organism arising from this zygote. This organism can in turn transmit the provirus to its descendants via the normal route of sexual reproduction. Activation of expression of the endogenous provirus in an animal (red dot) can lead to infectious spread throughout the body, viremia, and eventually leukemia (bottom). (B) The presence of endogenous retrovirus (ERV) genomes can be detected by probing the genomic DNA of a species using the DNA of an infectious retrovirus as the probe. Shown is a Southern blot (see Figure S4.3) of the ERV genomes present in the DNAs of a variety of mouse strains and subspecies. In this case, only the subclass of ERV genomes related to xenotropic murine retroviruses is being probed. Each band in a gel channel represents a restriction fragment in a cell genome that carries part or all of an ERV genome. The variability of ERV integration sites from one lab strain to another indicates that numerous novel ERVs have been integrated into the mouse germ line since the speciation of Mus musculus, from which all these strains derive. (C) In contrast to the mouse ERV genomes, those detected (using as a probe a fragment of the clone of a human ERV, above) in a collection of human DNAs show rather similar integration sites across the species, indicating their germ-line integration before the emergence of the human species; the polymorphic differences (black arrows) are largely the results of recombination between the pair of long terminal repeat (LTR) sequences at the ends of individual ERV proviruses and resulting deletion of the intervening stretches of proviral DNA. (B, from K. Tomonaga and J.M. Coffin, J. Virol. 73:43274340, 1999. C, from J.F. Hughes and J.M. Coffin, Proc. Natl. Acad. Sci. USA 101:16681672, 2004.)18 21. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Endogenous proviruses that are actively transcribed in an animals tissues may create a viremia and, following millions of replication cycles, may induce cancer in that animal. Such disease-inducing endogenous proviruses are therefore disadvantageous and, during the course of evolution, will be eliminated from a species gene pool. This explains why the endogenous proviruses found in the germ lines of most species are, with rare exception, either transcriptionally silent or incapable of encoding infectious virus particles. Careful examinations of the genomes of a variety of mammalian and avian species demonstrate the presence of numerous endogenous retroviral genomes, most of which are clearly relics of germ-line infections that occurred in the distant evolutionary past. Having resided for millions of years in a species germ line, most have suffered so many mutations that they are no longer able to specify infectious virus particles. However, a small subset of endogenous viral genomes, notably those that have been recently inserted into a species germ line, may remain genetically intact. Given the proper stimulus, these previously latent proviruses may suddenly be transcribed in one or another cell, generating virus particles that spread via infection from this cell throughout the body and eventually trigger some type of malignancy, usually of hematopoietic cells (see Section 2014 Garland Science3.11). For example, the high rate of leukemia in mice of the AKR strain is attributable to the frequent spontaneous activation of an endogenous murine leukemia provirus, the subsequent infectious spread of virus throughout the mouse, viremia, and, finally, via insertional mutagenesis, the activation of a protooncogene and the eruption of a leukemia. Such germ-line transmission of viral genomes was thought to be nothing more than a laboratory curiosity until reports, beginning in 2006, revealed that certain people carry loads of human herpesvirus type 6 (HHV-6) in their peripheral mononuclear cells (for example, lymphocytes, monocytes, macrophages) that are 1000 to 10,000 times higher than the average person carries. (Latent HHV-6 infections are widespread in the general population.) Subsequent studies revealed that these high loads of HHV-6 were often present in multiple members of a family and that in these families, HHV-6 genomes were inherited in the germ line integrated in certain chromosomes, specifically in telomeric DNA. Thus, a mechanism that HHV-6 usually employs in order to enter into a latent state (that is, by integrating its genome into one or another chromosome; see Section 3.6) occasionally results in germ-line transmission. The clinical consequences of inherited HHV-6 genomes remain unclear.19 22. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 4.2 Boveri and Hansemann independently hypothesized genetic abnormality as the cause of cancer cells malignant behavior The discovery that cancers arise as a consequence of aberrations in genetic material is widely attributed to the work of Theodor Boveri (18621915), a dominant figure in early twentieth-century German biology. This story, like many accounts of scientific history, is actually a bit more complicated and more interesting than conventional accounts depict. The roots of this notion actually reach back to an earlier set of observations by Boveris contemporary David Paul von Hansemann (1858 1920) (Figure S4.2A). In 1890, Hansemann, a pathologist, first reported his observations that the mitotic figures (for example, see Figure 12.38) of cancer cells contained a variety of abnormal chromosomal configurations, including multipolar mitotic spindles unusual arrays of chromosomes at anaphase, and asymmetric mitoses. He also proposed that the cells within tumors underwent partial dedifferentiation, another theme that anticipated twentiethcentury histopathological studies of tumors. Hansemann continued to report his observations in the decade that followed, but at the time had little insight into the cell-biological consequences of abnormal chromosome number and configuration. In 1900, the work of Mendel, long forgotten, was rediscovered by three geneticists. They popularized the idea that cells are essentially diploid, with pairs of genetic determinants being separated from one another during meiosis. In the several years that followed, the behaviors of Mendels genetic determinants were realized to parallel that of chromosomes, leading many to conclude that the chromosomes were the seat of heredity. Hence, cells with abnormal (A) 2014 Garland Sciencenumbers of chromosomes were perceived as being genetically mutant, though the term mutant had not yet come into currency. Unlike Hansemann, Boveri (see Figure S4.2B) was a zoologist and worked largely with early sea urchin embryos. He hardly acknowledged the work of Mendel and minimally that of Hansemann. Instead, his own work in manipulating metaphase figures in early sea urchin embryos led him to propose that the karyotypic aberrations observed occasionally in otherwise normal cells represented genetic abnormalities, that each chromosome carries a distinct subset of the genetic information present in the genome as a whole, and that the karyotypic abnormalities within cancer cells were responsible, in some fashion, for the aberrant behavior of cancer cells. The corrupted information content present in one cell was, he proposed, heritable from one cell generation to the next. Boveris posthumously published 1916 monograph, in which he laid out these ideas, was followed 13 years later by an English translation, carried out by his doting widow, the former Marcella OGrady of Boston, Massachusetts. Hansemann lacked a loyal widow and translator, and so awareness of his pioneering contributions in connecting an abnormal genetic constitution of cells with the development of tumors receded. Boveris personality also may have helped impress on the scientific community his unique role in many of these advances; an otherwise uncritical colleague described him as vehement, inflexible, and [a] relentless assailant. Today, few remember that Hansemann began the charge that led to the now widely embraced notion that cancer cells are genetically abnormal, and that these cells chromosomes are responsible for their malignant behavior.(B)Figure S4.2 The first clues to the genetic origins of human tumor development (A) David Paul von Hansemann described the abnormal mitotic figures and chromosomes in human cancer cells without knowing their significance. (B) Theodor Boveri drew on Hansemanns observations and his own to propose that abnormal chromosomes somehow were responsible for the abnormal behaviors of cancer cells. (A, courtesy of Wolfgang von Hansemann. B, courtesy of the Biocenter, University of Wzburg.)TBoC2 s4.0220 23. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 4.3 Southern and Northern blotting Southern blotting was invented in 1975 by the British biologist Edwin Southern as a means of detecting specific DNA fragments that are generated by restriction enzyme cleavage of DNA; these fragments may be present amid a millionfold excess of other concomitantly generated DNA fragments, revealing the sensitivity of this procedure (Figure S4.3). Within several years, the procedure was adapted to detect specific mRNA molecules, once again present amid a vast excess of unrelated molecules, in this case other mRNA species; for want of a better name, this(A)sresiphotro eleclabeled RNA or DNA of known sizes as size markersunlabeled RNA or DNA 2014 Garland Sciencenewer procedure was dubbed Northern blotting. Thereafter, a further adaptation of the procedure was developed in order to detect individual proteins amid a vast excess of unrelated proteins and termed the Western blotting procedure (not shown here). While the Southern and Northern blots use radiolabeled nucleic acid probes, the Western blotting procedure uses specific antibodies, usually monoclonal antibodies, to detect proteins that have been adsorbed to filters and for this reason is also termed immunoblotting.stack of absorptive paper towels (B)REMOVE NITROCELLULOSE PAPER WITH TIGHTLY BOUND NUCLEIC ACIDS(C)gelnitrocellulose paperagarose gelNUCLEIC ACIDS SEPARATED ACCORDING TO SIZE BY AGAROSE GEL ELECTROPHORESISsponge buffer SEPARATION OF NUCLEIC ACIDS BLOTTED ONTO NITROCELLULOSE PAPER BY SUCTION OF BUFFER THROUGH GEL AND PAPERFigure S4.3 Southern and Northern blotting procedures Use of these blotting procedures makes possible the detection of specific fragments of the cell genome (or specific RNA transcripts) if an appropriate radiolabeled DNA probe is available. (A) Either DNA fragments that have been generated by restriction enzyme cleavage of genomic DNA (in a Southern blot) or a mixture of cellular RNAs (in a Northern blot) are resolved by gel electrophoresis. (B) The gel slab is then placed beneath a nitrocellulose filter, and paper towels (or other absorptive material) are used to wick fluid through the gel, allowing the DNA (or RNA) molecules to adsorb to the filter paper, creating a replica of their previous positions in the gel. (C) The filter paper is peeled away from the gel and (D) placed in a plastic bag together with a solution of radiolabeled probe (pink). (E) Hybridization of the probe with complementary DNA (or RNA) molecules adsorbed on the filter and subsequent autoradiography with a photographic emulsion allow the detection of DNA fragments (or RNA molecules) that were present in the initial DNA (or RNA) preparation; these are manifested by bands of silver grains on the film. (From B. Alberts et al., Molecular Biology of the Cell, 5th ed. New York: Garland Science, 2008.)(D)LABELED PROBE HYBRIDIZED TO ADSORBED DNA OR RNA sealed plastic baglabeled probe in buffer LABELED PROBE HYBRIDIZED TO COMPLEMENTARY DNA OR RNA; BANDS VISUALIZED BY AUTORADIOGRAPHY (E)positions of labeled markerslabeled bandsTBoC2 b4.04/s4.0321 24. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 4.4 Genes undergo amplification for a variety of reasons The process of gene amplification is defined as a preferential increase in the copy number of a discrete chromosomal region (an amplicon), rather than increases in the ploidy of an entire genome, as may occur in certain specialized mammalian cells. In mammalian cells, gene amplification seems to occur only as a pathological process accompanying and driving tumor promotion (Figure S4.4). In other phyla,however, it may be part of a normal developmental program that responds to the need to express large amounts of a certain protein or group of proteins; this is achieved by directed, localized gene amplification, which results in proportional increases of the corresponding mRNA and protein. For example, in Drosophila, genes specifying chorion proteins undergo amplification in order to enable the synthesis of massive amounts of these proteins during oogenesis.ss nick or DSB(A)Alu elementsMYBMYB(B)strand invasion and homologous recombination with upstream repeat on sister chromatid MYBMYB(C) MYBMYB(D) MYBMYBMYB 2014 Garland ScienceFigure S4.4 Mechanism of generating tandemly duplicated genes A number of molecular mechanisms have been proposed to explain the generation of tandemly duplicated genes, such as those that form the homogeneously staining regions (HSRs) that create oncogene amplification. In the example presented here, the mechanism of amplification of the MYB oncogene via tandem duplication was studied in a human T-cell acute lymphoblastic leukemia. The amplification mechanism was traced, with high likelihood, to the presence of Alu elements, which are present in about one million copies scattered throughout the human genome (see Figure 4.7). In this case, the normal human MYB gene is flanked by two Alu repeat sequences. (A) When the MYB gene underwent replication during the S phase of the cell cycle, a single-strand (ss) nick or doublestrand break (DSB) in the newly synthesized upper sister chromatid allowed the resulting free end (which contains an Alu sequence, empty rectangle) to invade the doublestrand helix of the lower sister chromatid and anneal to another Alu sequence (dark red rectangle) that lies upstream of the MYB gene on the lower sister chromatid (B). The 3 end of the newly synthesized, annealed strand could then be elongated using as a template this lower sister chromatid (light blue). (C). Subsequently, this elongating strand could jump back to the upper sister chromatid (dark green) and continue elongation, generating two head-to-tail tandem copies (D) of the MYB gene. This process might then be repeated, leading to an exponential expansion of this chromosomal region. (From J. ONeil et al., J. Exp. Med. 204:30593066, 2007.)TBoC2 s4.0422 25. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 5.1 Making anti-Src antibodies presented a major challenge Thorough biochemical analyses of thousands of cellular proteins have depended upon the ability to detect and isolate a protein of interest (for example, Src) amid the complex soup of proteins present in cells. This detection frequently relies on the use of antibodies that specifically recognize and bind a protein such as Src while ignoring all other cellular proteins around it. The production of such antibodies involves the injection of an intact protein (for example, the entire Src protein) or a chemically synthesized oligopeptide (for example, a short protein whose amino acid sequence reflects the sequence of a small segment of the Src protein) into an animal, usually a mouse, rabbit, or goat. The hope is that the injected material will be immunogenic (will elicit an immune response) in the animal, causing it to form antibodies that specifically bind and precipitate this protein from a cell lysate (a preparation of disrupted cells). The immune systems of mammals often react vigorously to a foreign antigen, that is, a protein carrying peptide sequences that are unfamiliar. Conversely, the immune system often mounts no immune response at all (and thus fails to make antibody) against an injected protein that closely resembles one of the proteins normally present in the animal. This lack of immunoreactivity against familiar proteins is termed immunological tolerance, a phenomenon that we will return to in Chapter 15. Those who first produced anti-Src antibodies used a strain of RSV that can infect and transform mammalian cells and can therefore induce tumors in mammals. They anticipated that the resulting tumors would express the chicken Src protein, and that dying tumor cells would expose this protein to the immune systems of the tumor-bearing mammalian hosts and thus provoke an immune response. In fact, the Src protein proved to be poorly immunogenic in most animals. The reason for this was discovered much later: the structure of the Src protein is highly conserved evolutionarily. Accordingly, the chicken Src protein is almost identical to the corresponding protein of most mammalian species and is therefore not readily recognized as a foreign protein by these species immune systems. In the end, these investigators succeeded (for unknown reasons) in producing anti-Src antiserum only from rabbits bearing RSVinduced tumors. The factors that determine the immunogenicity of a protein such as Src in one or another species are complex and often obscure. Once in hand, the anti-Src antiserum could be used to precipitate (that is, immunoprecipitate) Src protein from lysates of a wide variety of RSV-transformed cells and from normal, untransformed cells (Figure S5.1). As in many such analyses, these lysates were prepared from cells that had 2014 Garland Sciencebeen incubated in the presence of radiolabeled amino acids, resulting in the incorporation of radiolabel in the cells proteins. Following immunoprecipitation, the resulting proteins were resolved by gel electrophoresis, and the presence of radiolabeled proteins was detected by autoradiography. In the present case, the long-sought Src protein was detected as a polypeptide migrating as a 60-kD protein.150,00088,000Pr76 Pr66 60,000 Pr5335,000 p27 (daltons mol. wt.)p19 T7123456789p12, 15Figure S5.1 Immunoprecipate of Src protein Initially, the only means of studying the Src protein depended on first immunoprecipitating it from the mixture of tens of thousands of other cellular proteins. Many attempts at making an anti-Src serum finally resulted in an antibody preparation that could be used to immunoprecipitate this protein from cells. Molecular weight markers are in the leftmost channel T7; channels 13: lysate of normal, uninfected cells; channels 46: lysate of cells infected with an RSV mutant with a deleted src gene; channels TBoC2 b5.06/s5.01 79: lysate of cells infected with wild-type RSV. Channel 7: normal rabbit serum; channel 8: tumor-bearing rabbit serum; channel 9: same as channel 8 except pre-incubated with lysate of RSV particles. The band at 60 kD in channels 8 and 9 (arrow) represented the first detection of the RSV oncoprotein. (p12, p15, p19, p27, Pr53, Pr66, and Pr76 are RSV virion proteins.) (From A.F. Purchio et al., Proc. Natl. Acad. Sci. USA 75:15671571, 1978.)23 26. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 5.2 The protozoan roots of metazoan signaling In the Cambrian era, ~540 million years ago, a diverse array of complex multicellular organisms evolved; many of these were the ancestors of contemporary metazoan phyla and already had recognizable features of modern organisms. Examination of contemporary animal phyla leads to a key insight into our origins: a metazoan organization plan was invented only once, and all modern metazoa descend from a common pre-metazoan ancestor that somehow solved the problem of cellcell communication and the organization of complex tissues. This organization depended in significant part on the evolution of intercellular signaling systems, including the growth factors, receptors, and cellcell adhesion molecules that are found in all modern metazoa. The fossil record indicates that during the earlier Vendian/Ediacaran era, which spanned from 650 to 540 million years ago, a variety of soft-bodied metazoa were already present, pushing the evolutionary origins of metazoa back another ~100 million years. The nature of the common pre-metazoan ancestor has been obscure until recently. However, accumulating evidence now implicates choanoflagellates as being the closest living relatives of metazoans. Each single-cell form of a choanoflagellate has a(A) 2014 Garland Sciencesingle flagellum (which provides propulsion) that is surrounded by a large collar (assembled from 3040 microvilli) that enables these protozoans to trap the bacteria that they consume for food (Figure S5.2). Large multicellular choanoflagellate colonies contain flagellated cells on the outside and possibly other flagellum-free cell types on the inside. The evolution of these multicellular colonies presumably depended on the development of cellcell signaling and adhesion molecules. Importantly, sequencing of a choanoflagellate genome indicates that these protozoans make proteins that are found in higher metazoa, including receptor tyrosine kinases, EGFlike proteins, G-proteincoupled receptors, a TNF receptor-like molecule (Chapter 9), SH2 groups (Chapter 6), and cadherins (which bind metazoan epithelial cells together in two-dimensional sheets; Chapter 13). Once evolved to enable the assembly of these primitive multicellular colonies, these proteins could then be adapted in various ways to permit the formation and explosive diversification of far more complex multicellular animals during the early Cambrian period. At the same time, the signaling systems that were assembled from these proteins laid the foundations for the disease that, half a billion years later, we call cancer.(B)5 mFigure S5.2 Origins of cellcell communication The tyrosine kinase receptors, along with a series of other important signaling molecules, were clearly developed long before the Cambrian explosion generated the ancestors of the various modern metazoan phyla. The choanoflagellate genome encodes many of these critical metazoan signaling molecules. (A) The choanoflagellates received their name from the distinctive collar that surrounds their single flagellum and is composed of an array of actin-rich microvilli (red). The single flagella, like the microtubules of the cytoplasm, are revealed here by an anti-tubulin antibody (green), while the nuclei are stained blue. (B) This scanning electron micrograph reveals that each solitary cell extends a single long flagellum with a surrounding collar that, in this case, has been disrupted into its individual component microvilli. Bacteria, which are trapped by these microvilli and used as food by the choanoflagellates, are seen nearby. (A, courtesy of Melissa TBoC2 n5.108,9/s5.02 Mott; from N. King, Curr. Biol. 16:R113114, 2004. B, courtesy of M.J. Dayel, K. McDonald, and N. King.)24 27. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 5.3 Lateral interactions of cell surface receptors While GF receptors, such as those responding to EGF or PDGF, and integrins have been presented here as independently acting signaling proteins, it is becoming increasingly apparent that these cell surface receptors often form lateral complexes with one another and regulate each others signaling. In addition, integrin:RTK complexes are formed through association of these proteins with components of the ECM (Figure S5.3). For example, after binding its extracellular matrix (ECM) ligands, the v1 integrin can cause phosphorylation of the EGF receptor even in the absence of EGF or EGFrelated ligands. This cross-phosphorylation appears to require physical association between the integrin and the EGF-R and depends, as well, on the complex of signaling molecules that the integrin attracts to its C-terminal tail in the cytoplasm. The Src tyrosine kinase, an important component of these integrin signaling complexes (see Figure 6.24) is activated following v1 integrin binding to the ECM and is required for the phosphorylation of tyrosine residues in the C-terminal tail of the closely apposed EGF-R. This phosphorylation occurs independently of the EGF-Rs binding its normal ligands and results in a different spectrum of C-terminal phosphotyrosines than when the EGF-R binds its ligands. In addition, the amount of EGF-R present on the cell surface increases after the v1 integrin binds ECM ligands. Indeed, efficient EGF-R signaling may require integrin association and integrin-mediated signaling. This example is only one of many. Thus, integrin-mediated modulation of tyrosine kinase receptor (RTK) signaling has been described for the platelet-derived growth factor- (PDGF-) receptor; a vascular endothelial growth factor (VEGF) receptor; the hepatocyte growth factor (HGF) receptor, termed Met, andthe Met-related Ron RTK, which binds HGFL (HGF-like) ligand; the insulin receptor (IR); and the EGF-Rrelated ErbB2/HER2 receptor. And not unexpectedly, interactions between integrins and RTKs can be reciprocal: activation of RTKs by their respective ligands can stimulate signaling by the proteins associated with the C-terminal tails of integrins. Such reciprocal signaling suggests that RTKs signal in part by activating integrin-associated signaling proteins, while integrins signal in part by activating RTK-associated proteins. As receptor biochemistry is studied in increasing detail, it is also apparent that cross-phosphorylation between receptors occurs commonly and may represent a critical mechanism for controlling cell typespecific signaling. Within the EGF-R family of receptors, for example, HER2 can form heterodimeric complexes with HER3 and phosphorylate the C-terminal tail of the latter; in fact, HER3 itself lacks a catalytically functional kinase. Perhaps more