4268 Lectures 18-19 Study Guide PDF

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Lectures 18-19 Study Guide – Metastasis 1) What are micrometastases? (Pg. 642, paragraph 4) In a variety of human tumor types, the dissemination of cancer cells throughout the body has already occurred by the time a primary tumor is first detected; at the time of initial diagnosis, these scattered cells may be inapparent because they form only minute tumor colonies—micrometastases. Such behavior provokes a question that we will confront in this chapter and again in Chapter 16: Do the properties of a primary tumor reveal whether it has broadcast cancer cells throughout the body that will even- tually create life-threatening metastatic disease long after the primary tumor has been surgically removed? 2) Describe the six distinct steps of the invasion-metastasis cascade. (Pg. 644, Figure legend 14.3) Figure 14.3 The invasion–metastasis cascade This depiction of the invasion– metastasis cascade ascribes seven distinct steps to the overall process. The initial step of localized invasiveness enables in situ carcinoma cells to breach the basement membrane. Thereafter, they may intravasate into either lymphatic or blood microvessels. The latter may then transport these cancer cells, via the general circulation, to distant anatomical sites, where they may be trapped, subsequently extravasate, and form dormant micrometastases. Some of the micrometastases may eventually acquire the ability to colonize the tissue in which they have landed, enabling them to form a macroscopic metastasis. The last step—colonization—seems to be the most inefficient of all. The small probability of successfully completing all steps of this cascade explains the low likelihood that any single cancer cell leaving a primary tumor will succeed in becoming the founder of a distant, macroscopic metastasis. More recent molecular and cellbiological studies suggest an alternative depiction involving two major phases: the first enables the physical dissemination of cancer cells from the core of the primary tumor to the parenchyma of a distant tissue, while the second—colonization—depends on the adaptation of disseminated cancer cells to the microenvironment of this tissue. 3) How are metastasizing tumor cells proposed to survive the fluid forces of the circulatory system? (Pg. 646, paragraph 2, sentence 3) Some experi- mental models of metastasis in the mouse provide clear indication that the survival of metastasizing cancer cells in the general circulation is greatly enhanced if they can attract an entourage of blood platelets to escort them through the rapids into safe pools within tissues. 4) Describe in detail the two final steps in metastasis: extravasation and colonization. (Pg. 650, paragraph 1, sentences 2-6; Pg. 652, paragraph 1, sentences 2-3) COLONIZATION: TUMOR CELLS ABLE TO FIND THEMSELVES IN A TISSUE THAT WILL PROVIDE THE PROPER GROWTH FACTORS THAT WILL FEED THAT PARTICULAR CELL TYPE AND ONE OF THE MOST IMPORTANT THINGS

IS GROWTH FACTORS TO ALLOW FOR CELL DIVISION AND VASCULARIZATION- VEGF EXTRAVASATION: INVOLVES EXPRESSING PROTEANASES TO ALLOW TUMOR CELLS TO LEAVE THE VESSEL PROPER Cancer cells can use several alternative strategies to extravasate. They may proceed immediately to elbow their way through the vessel wall (Figure 14.9A–C). Their abil- ity to do so may depend on the same biochemical and cell-biological mechanisms that previously enabled them or their immediate ancestors to invade from the primary tumor and to intravasate (see, however, Sidebar 14.1). Alternatively, they may begin to proliferate within the lumen of the vessel, creating a small tumor that grows and eventually obliterates the adjacent vessel wall (see Figure 14.9D). In doing so, they push aside endothelial cells, pericytes, and smooth muscle cells that previously sepa- rated the vessel lumen from the surrounding tissue, the latter often being called the tissue parenchyma. Interestingly, close associations of disseminated cancer cells and macrophages have been documented at sites of extravasation, suggesting that, as is the case with intravasation (see Figure 14.7), cancer cells recruit macrophages to help them escape from the circulation into the tissue parenchyma. This last step is also a chal- lenging one—perhaps the most difficult step of all, ostensibly because the foreign tis- sue environments encountered by the newly arrived cells do not provide them with the collection of familiar growth and survival factors that allowed their progenitors to thrive in the primary tumor site. Without these various types of physiologic support, the metastasizing cells may rapidly die or, at best, survive for extended periods of time as micrometastases that can only be detected microscopically and rarely increase beyond this size. 5) How are micrometastases detected? (Pg. 652, paragraph 2) Antibodies reactive with cytokeratins are useful for detecting the micrometastases that primary carcinomas spawn in the bone marrow and blood, while an anti- body against the epithelial cell adhesion molecule (EpCAM) is often used to detect micrometastases in the lymph nodes. In all these cases, the presence of isolated cytokeratin-positive (and thus epithelial) cells in otherwise fully mesenchymal tissues represents a clear sign that metastatic seeding has taken place. Current microscopic techniques using cytokeratin-specific antibodies make it possible to detect a single-cell micrometastasis among 105 or even 106 surrounding mesenchymal cells in the blood, bone marrow, or lymph node (Figure 14.10A and B). Slightly larger micrometastases can often be detected in the lymph nodes that are connected with a primary tumor via draining lymphatic ducts 6) What is the critical change a tumor cell must undergo in order to become motile and invasive? (Pg. 657, paragraph 2, sentences 1-2; Pg. 659, paragraph 2) In order to acquire motility and invasiveness, carcinoma cells must shed many of their epithelial phenotypes, detach from epithelial sheets, and undergo a drastic alteration—the epithelial–mesenchymal transition (EMT), which was mentioned in the context

of wound healing (see Section 13.3). Recall that an EMT involves a shedding by epithelial cells of their characteristic morphology and gene expression pattern and the assumption of a shape and transcriptional program characteristic of mesenchy- mal cells. The normal and pathological versions of the EMT involve, in addition to changes in shape and the acquisition of motility, fundamental alterations in the gene expression profiles of cells (Table 14.2). Expression of E-cadherin and cytokeratins—hallmarks of epithelial cell protein expression—is repressed, while the expression of vimentin, an intermediate filament component of the mesenchymal cell cytoskeleton, is induced (Figure 14.15). Epithelial cells that have undergone an EMT often begin to make fibronectin, an extracellular matrix protein that is normally secreted only by mesenchymal cells such as fibroblasts. At the same time, expression of a typical fibroblastic marker—N-cadherin—is often acquired in place of E-cadherin. 7) How does E-cadherin expression influence the epithelial cell phenotype? (Pg. 659, paragraph 3, sentences 3-4; entire paragraph 4 and paragraph 6) In normal epithelia, the ectodomains of E-cadherin molecules extend from the plasma membrane of one epithelial cell to form complexes with other E-cadherin molecules protruding from the surface of an adjacent epithelial cell. This enables homodimeric (and higher-order) bridges to be built between adjacent cells in an epithelial cell layer, resulting in the adherens junc- tions that are so important to the structural integrity of epithelial cell sheets. The cytoplasmic domains of individual E-cadherin molecules are tethered to the actin fibers of the cytoskeleton via a complex of α- and β-catenins (see Figure 14.14D) and other ancillary proteins. The actin cytoskeleton, for its part, provides tensile strength to the cell. Hence, by knitting together the actin cytoskeletons of adjacent cells, E-cadherin molecules help an epithelial cell sheet resist mechanical forces that might otherwise tear it apart. Once E-cadherin expression is suppressed, many of the other cellphysiologic changes associated with the EMT seem to follow suit. Some experiments indicate that simply by suppressing the expression of the E-cadherin protein, cells acquire a mesenchymal morphology and increased motility. Additionally, in studies of several types of carcinoma cells that had lost Ecadherin expression, re-expression of this protein (achieved experimentally by introduction of an E-cadherin expression vector) strongly suppressed the invasiveness and metastatic dissemination of these cancer cells. Together, these diverse observations indicate that E-cadherin levels are key determinants of the biological behavior of epithelial cancer cells and that the cell-to-cell contacts constructed by E-cadherins impede invasive- ness and hence metastasis. 8) Traditional histopathological techniques do not demonstrate epithelial cells undergoing the epithelial-mesenchymal transition (EMT) at the invasive edges of primary carcinomas. Why? (Pg. 666, paragraph 3, sentences 1-2) In fact, traditional histopathological techniques have failed to demonstrate the EMT at the invasive edges of primary carcinomas for a simple reason: once tumor cells

undergo a full EMT (that is, shed all epithelial traits and acquire mesenchymal ones instead), they are essentially indistinguishable from the mesenchymal cells in the surrounding stroma (Supplementary Sidebar 14.5). For this reason, demonstration of an EMT at the invasive edges of tumors has required the use of antibodies and cellular reagents that are not normally used in diagnostic pathology laboratories.

9) A. Experimental evidence suggests that a collection of contextual signals induce the EMT in adjacent carcinoma cells. What are these signals? (Pg 666, paragraph 6) To date, far more evidence has accumulated pointing to active heterotypic signaling between the stromal and epithelial compartments within carcinomas. Abundant evi- dence indicates that TGF-β is an important agent for conveying these stromal signals. Other observations implicate a variety of other factors, including Wnts, TNF-α (tumor necrosis factor-α), epidermal growth factor (EGF), HGF (hepatocyte growth factor), IGF-1 (insulin-like growth factor-1), and prostaglandin E2 (PGE2). Some research even implicates direct contact of carcinoma cells with collagen type I, which is present in abundance in the stroma but absent in the epithelial compartment of the tumor. It appears likely that these stromal signals act in various combinations to induce epithelial cells to activate their previously latent EMT programs. B. A diagram summarizing contextual signals known to be important in the EMT is shown in Figure 14.24. How was NF-kB signaling found to be critical for the EMT? (Pg. 668, paragraph 4, sentences 2-6) TGF-B BINDING TO NFKB PATHWAY HELPING TO CONTRIBUTE TO EMT For example, TNF-α act- ing in concert with TGF-β appears to be effective in inducing an EMT. Early in tumor progression, TNF-α is often produced by inflammatory cells, such as macrophages (see Section 11.16); it can then function via its receptor to activate the NF-κB signaling pathway in epithelial cells. TGF-β also activates the NF-κB pathway in epithelial cells, such as the immortalized mouse mammary epithelial cells discussed above. In vari- ous tumors, TNF-α and TGF-β may contribute to the long-term maintenance of active NF-κB signaling. This signaling seems to be critical for the induction and maintenance of an EMT, since inhibition of NF-κB signaling prevents expression of the EMT pro- gram. 10) The EMT is controlled through transcription factors that play key roles in the gastrulation phase of embryogenesis. What type of transcription factors are Snail and Slug (cool names) and what is their role in the EMT? (Pg. 675, paragraph 4) Snail and Slug (sometimes called SNAI1 and SNAI2) are members of the C2H2type zinc finger TFs. The Snail–Slug TFs seem to operate largely as repressors of transcription. Thus, both have been found to be able to repress transcription of the E-

cadherin gene. As we read earlier, the loss of E-cadherin expression can, on its own, cause epithelial cells to assume many of the phenotypic changes associated with an EMT. 11) A. What effect does Twist (another cool name) have on the phenotype of a tumor cell? (Pg. 684, paragraph 1, sentence 2) BY Slug has also been implicated in the repression of E-cadherin expression in human breast cancers. And significantly, both Twist and Slug enable cells to resist apoptosis and anoikis, and therefore can protect disseminating cells from some of the physiologic stresses that would normally cause their death long before they reach distant tissue sites and form micrometastases. B. How does Twist expression affect the prognosis of melanoma patients? (Pg. 684, Figure 14.31B) LOW TWIST LEVELS IN THE MELANOMA TISSUE THAT PTS HAD A BETTER SURVIVAL RATE COMPARED TO PTS WITH HIGH LEVELS OF TWIST - TWIST ALLOWS FOR APOPTOSIS AND ALLOWS FOR DOWN REGUALTION OF E-CADHERIN (B) Sections of primary melanomas were immunostained for Twist expression and correlated retrospectively with the long-term survival of the patients bearing these tumors 12) What is the role of membrane type-1 matrix metalloproteinase (MT1-MMP) in metastasis? (Pg. 686, paragraph 2) The activities of MT1-MMP, which plays the leading role in BM breakdown, seem to be confined through its concentration at discrete cell surface foci, initially termed podosomes but increasingly called invadopodia because of the involvement of these structures in cancer cell invasion (Figure 14.33). Early in malignant progres- sion, MT1MMP displayed on the surface of carcinoma cells can cleave collagen type IV, the collagenTthBoaCt2imbp1a4r.ts31ri/g1i4d.it3y2to the basement membrane (BM). The resulting weakening of the BM allows cancer cells to begin invading the underlying stroma (see Figure 14.4). Once in the stroma, an invading carcinoma cell confronts a dense net- work of cross-linked collagen type I fibers that obstructs further advance; here once again, MT1-MMP plays a central role. MT1-MMP initiates collagen I degradation and then calls in an inactive pro-enzyme (pro-MMP-2) of stromal origin, which it activates by cleavage. The resulting active MMP-2 then operates in the peri-cellular space to further cleave collagen I into lower–molecular-weight fragments. Without these steps, the dense networks of collagen I fibers that are present in the stromal extracellular matrix block cancer cell invasion 13) The following questions refer to cell motility:

A. What is the role of hepatocyte growth factor (HGF) in epithelial cell motility? (Pg. 689, paragraph 4, sentences 3-4) In the case of epithelial cells, the best inducer of motility is usually hepatocyte growth factor (HGF); this protein is also called scatter factor (SF) in recognition of its ability to induce multidirectional movement of cells in monolayer culture. Many types of epithelial cells express Met, the receptor for HGF, and such cells have been found to acquire motility in response to HGF treatment (see Figure 14.23A). Similarly, EGF is clearly able to induce motility of breast cancer cells (see Figure 14.22B). B. Describe the basic steps of cell locomotion. (Pg. 690, Figure legend 14.36) Figure 14.36 Locomotion of cells on solid substrates The locomotion of a cultured cell depends on the coordination of a complex series of changes in the cytoskeleton, as well as the making and breaking of focal contacts with the underlying solid substrate. The cell organizes actin fibers in order to extend lamellipodia at its advancing/leading edge and to establish new focal contacts. At the same time, stress fibers, also consisting of actin, are used to contract the trailing edge of the cell, where focal contacts are being broken. The making and breaking of the focal contacts depend on localized modulation of the affinities of various integrins for extracellular matrix (ECM) components, represented here by the yellow substrate. C. What is the role of the Rho GTPase family in cell motility? (Pg. 690, paragraph 3, sentences 1-2, 4-6; Pg. 691, paragraph 1, sentence 2; Pg. 693, paragraph 3, sentence 3; paragraph 4, sentences 2, 4-5;Pg. 694, paragraph 1) RHO GTPase would definitely be functioning in the carcinoma cells The detailed management of cell shape and motility is under the control of members of a group of Ras-related proteins belonging to the Rho family. As discussed briefly in Chapter 6, the Rho proteins, like Ras, operate as binary switches, being in a function- ally active state while binding GTP and in an inactive state once they hydrolyze their bound GTP to GDP. They are divided into three subfamilies—the Rho proteins proper, the Rac proteins, and Cdc42. Like the Ras proteins, most members of the Rho protein family bear lipid groups at their C-termini that enable anchoring to intracel- lular membranes. Each of these has specialized functions in reorganizing cell shape and enabling cell motility This focused activation seems to depend, in turn, on the subcellular localization of specialized Rho GEFs (guanine nucleotide exchange factors), which, like Sos (see Section 6.2), operate to convert Rho proteins from their inactive GDP-bound form to their active GTP-bound form; there are about 80 Rho GEFs in mammalian cells, few of which have been studied in any detail. The alternative to localized activation—global activation—would lead to attempts by a cell to move simultaneously in all directions, a scenario suggested by the lamellipodia of Figures 14.37C and 14.39C, which form a continuous ring around the entire perimeter of the cytoplasm. (The global activation of

Rac function in the cell depicted in Figure 14.39C is an artifact of introducing mutant, constitutively activated Rac protein into the cell by micro-injection.) By activating Rac proteins, the Tiam1 GEF encourages the localized polymerization of actin at the leading edge of migrating cells, thereby yielding the lamellipodia that are so critical to cell locomotion For example, Rho proteins like RhoA and RhoB, act- ing in concert with Rac proteins, promote the establishment of new points of adhesion between the leading edge of the cell and the extracellular matrix. Rac and Cdc42 proteins also appear able to induce expression of certain secreted proteases, notably the matrix metalloproteinases described in the last section. By doing so, they may coordinate localized remodeling of the extracellular matrix with extension of lamellipodia at the leading edge of a motile cell. The contraction of the cell body (which helps to pull the lagging edge of the cell forward toward the leading edge; see Figure 14.36) is equally important for a cell’s directed movement. This contraction is also governed largely by members of the Rho subfamily of proteins. By encouraging the formation of actin bundles in the cytoplasm, Rho pro- teins are able to create the structures known as “stress fibers” (see Figure 14.39B) and thereby contribute to the regulation of the contractility of the cytoplasm.

14) A. What is metastatic tropism? (Pg. 700, figure legend 14.43, sentence 3) In some cases, a tumor’s tendency to spawn metastases in one or another tissue reflects the abilities of the cancer cells from the primary tumor to adapt to (and thus colonize) the microenvironment of distant tissues; this likely explains the strong tendencies of prostate and breast cancers to generate metastases in the bone marrow. B. Describe the factors that control metastatic tropism. (Pg. 701, paragraph 3, sentence 2; Pg. 702, paragraph 2) Instead, in certain cases, the predilection to metastasize to a certain target organ is likely to be dictated by the layout of the ves- sels connecting the site of a primary tumor and the site of metastasis. The same logic may explain why breast cancer cells often form metastases in the lungs. As is the case with metastasizing colorectal carcinoma cells, wandering mammary carcinoma cells may not find that the lungs provide them with an especially hospita- ble environment, and individual cancer cells will have a low probability of successfully colonizing the lungs. Nonetheless, some m...