Chapter 7 Neoplasia: Molecula Basis of Cancer Flashcards

Question

Explain the Limitless replicative potential:

Answer 1

Tumor cells **have unrestricted proliferative capacity,** **avoiding cellular senescence and mitotic catastrophe.**

Answer 2

True Sustained angiogenesis Hence, tumors must induce angiogenesis.

Answer 3

True Defects in DNA repair

Answer 4

FIGURE 7-25 Flowchart depicting a simplified scheme of the molecular basis of cancer.

Answer 5

Genes that **promote autonomous cell growth in cance**r cells are called oncogenes

Answer 6

Genes that promote autonomous cell growth in cancer cells are called oncogenes, and their **unmutated cellular counterparts** are called proto-oncogenes

Answer 7

Oncogenes are created by mutations in proto-oncogenes and are characterized by the ability to promote cell growth in the absence of normal growth-promoting signals. **Their products, called oncoproteins**, **resemble the normal products of proto-oncogenes except that oncoproteins are often devoid of important** **internal regulatory elements,** and their production in the transformed cells does not depend on growth factors or other external signals. In this way cell growth becomes autonomous, freed from checkpoints and dependence upon external signals.

Answer 8

* The binding of a growth factor to its specific receptor * Transient and limited activation of the growth factor receptor, which, in turn, activates several signal-transducing proteins on the inner leaflet of the plasma membrane * Transmission of the transduced signal across the cytosol to the nucleus via second messengers or by a cascade of signal transduction molecules * Induction and activation of nuclear regulatory factors that initiate DNA transcription * Entry and progression of the cell into the cell cycle, ultimately resulting in cell division

Answer 9

* growth factors * or their receptors, * signal transducers, * transcription factors, * or cell cycle components.

Answer 10

Protooncogene: Mode of Activation :Associated Human Tumor **SIS (official name PBGFB)**:Overexpression:**Astrocytoma Osteosarcoma**

Answer 11

Protooncogene: Mode of Activation :Associated Human Tumor **HST1**:Overexpression:**Stomach cancer** **INT2 (official name FGF3):**Amplification:**Bladder cancer,Breast cancerMelanoma**

Answer 12

Protooncogene: Mode of Activation :Associated Human Tumor **TGFA**:Overexpression:**Astrocytomas, Hepatocellular carcinomas**

Answer 13

Protooncogene: Mode of Activation :Associated Human Tumor **HGF:** Overexpression: **Thyroid cancer**

Answer 14

Protooncogene: Mode of Activation :Associated Human Tumor **ERBB1(EGFR), ERRB2:** Overexpression: **Squamous cell carcinoma of lung, gliomas**

Answer 15

Protooncogene: Mode of Activation :Associated Human Tumor **FLT3:** Amplification: **Breast and ovarian cancers**

Answer 16

Protooncogene: Mode of Activation :Associated Human Tumor **RET** :Point mutation: **Leukemia**

Answer 17

Protooncogene: Mode of Activation :Associated Human Tumor **RET:**Point mutation: **Leukemia**

Answer 18

Protooncogene: Mode of Activation :Associated Human Tumor **RET**:Point mutation **:Multiple endocrine neoplasia 2A and B, familial medullary thyroid carcinomas**

Answer 19

Protooncogene: Mode of Activation :Associated Human Tumor **PDGFRB** Overexpression,translocation: **Gliomas, lekemias**

Answer 20

Protooncogene: Mode of Activation :Associated Human Tumor **KIT**: Point mutation: **Gastrointestinal stromal tumors, seminomas, leukemias**

Answer 21

Protooncogene: Mode of Activation :Associated Human Tumor **KRAS:** Point mutation: **Colon, lung, and pancreatic tumors**

Answer 22

Protooncogene: Mode of Activation :Associated Human Tumor **HRAS :**Point mutation: **Bladder and kidney tumors**

Answer 23

Protooncogene: Mode of Activation :Associated Human Tumor **NRAS:** Point mutation :**Melanomas, hematologic malignancies**

Answer 24

Protooncogene: Mode of Activation :Associated Human Tumor **ABL** :Translocation: **Chronic myeloid leukemia Acute lymphoblastic leukemia**

Answer 25

Protooncogene: Mode of Activation :Associated Human Tumor **BRAF** :Point mutation: **Melanomas**

Answer 26

Protooncogene: Mode of Activation :Associated Human Tumor **β-catenin:** Point mutation,Overexpression **:Hepatoblastomas, hepatocellular carcinoma**

Answer 27

Protooncogene: Mode of Activation :Associated Human Tumor **C-MYC:** Translocation: **Burkitt lymphoma**

Answer 28

Protooncogene: Mode of Activation :Associated Human Tumor N-MYC: Amplification**: Neuroblastoma, small-cell carcinoma of lung**

Answer 29

Protooncogene: Mode of Activation :Associated Human Tumor **L-MYC**: Amplification: **Small-cell carcinoma of lung**

Answer 30

Protooncogene: Mode of Activation :Associated Human Tumora **Cyclin D** :Translocation :**Mantle cell lymphoma**

Answer 31

Protooncogene: Mode of Activation :Associated Human Tumor **Cyclin D**: Amplification: **Breast and esophageal cancers**

Answer 32

Protooncogene: Mode of Activation :Associated Human Tumor **Cyclin E** :Overexpression: **Breast cancer**

Answer 33

Protooncogene: Mode of Activation :Associated Human Tumor * *CDK4** :Amplification or point mutation: * *Glioblastoma, melanoma, sarcoma**

Answer 34

The oncogenic versions of these receptors are **associated with constitutive** **dimerization and activation without binding to the growth factor** . Hence, the mutant receptors **deliver continuous mitogenic signals to the cell, even in the absence of growth factor in the** environment.

Answer 35

mutations, gene rearrangements, and overexpression

Answer 36

a **receptor tyrosine kinase**, exemplifies oncogenic **conversion via mutations and** **gene rearrangements.** [33]

Answer 37

The RET protein is a receptor for the **glial cell line–derived** * *neurotrophic factor and structurally related proteins that promote cell survival during neural** * *development**. RET is **normally expressed in neuroendocrine cells,** such as **parafollicular C cells** **of the thyroid, adrenal medulla, and parathyroid cell precursors.**

Answer 38

* *dominantly inherited MEN types 2A and 2B and familial** * *medullary thyroid carcinoma** ( Chapter 24 ).

Answer 39

**point mutations** in the **RET extracellular domain**cause constitutive dimerization and activation, leading to**medullary thyroid carcinomas and adrenal and parathyroid tumors.** **MEN 2A loves to TAP**

Answer 40

In MEN-2B, point mutations in the RET cytoplasmic catalytic domain alter the substrate specificity of the tyrosine kinase and **lead to thyroid and** **adrenal tumors *without involvement of the parathyroid.*** ***"MEN 2B- BONELESS"*** In all these familial conditions, the affected individuals inherit the RET mutation in the germline. Sporadic medullary carcinomas of the thyroid are associated with somatic rearrangements of the RET gene, generally similar to those found in MEN-2B.

Answer 41

* *Point mutations in FLT3**, the **gene encoding the FMS-like tyrosine kinase** * *3 receptor**, that **lead to constitutive signaling have been detected in *myeloid leukemias.***

Answer 42

eceptor tyrosine kinase c-KIT or PDGFR which are the **receptors for stem cell factor and PDGF,** respectively. These mutations are **amenable to specific inhibition by the tyrosine kinase inhibitor imatinib mesylate**. This type of therapy, directed to a specific alteration in the cancer cell, is **called targeted therapy .**

Answer 43

epidermal growth factor (EGF) receptor family

Answer 44

The normal form of ERBB1, the EGF receptor gene, is **overexpressed in up to 80% of squamous cell carcinomas of the lung**, in**50% or more of glioblastomas** ( Chapter 28 ), and in **80% to 100% of head and neck tumors.** [38,] [39]

Answer 45

Likewise, the **ERBB2 gene (also called HER-2/NEU**), the**second member of the EGF receptor**family, is**amplified in approximately 25%** **of breast cancers** and in **human adenocarcinomas** arising **within the ovary, lung, stomach, and salivary glands.** [36] GLANDS!!! Because the molecular alteration in **ERBB2 is specific for the cancer cells,** **new therapeutic agents consisting of monoclonal antibodies specific to ERBB2 have been developed and are currently in use clinically, providing yet another example of targeted** therapy.

Answer 46

* *RAS** family of guanine triphosphate (GTP)-binding proteins (G proteins) .

Answer 47

HRAS, KRAS, NRAS)

Answer 48

Point mutation of **RAS family genes** is the single most common abnormality of proto-oncogenes in human tumors

Answer 49

**90% of pancreatic adenocarcinoma**s and cholangiocarcinomas contain a **RAS point mutation,** as do about **50% of colon, endometrial, and thyroid cancers and 30% of lung adenocarcinomas** **and myeloid leukemias**

Answer 50

In general, carcinomas : * (particularly from colon and pancreas) have mutations of **KRAS** * bladder tumors have **HRAS** mutations, * hematopoietic tumors bear **NRAS** mutations. RAS mutations are infrequent in certain other cancers, such as those arising in the uterine cervix or breast

Answer 51

RAS plays an **important role in signaling cascades downstream of growth factor receptors, resulting in mitogenesis.** For example, abrogation of RAS function blocks the proliferative response to EGF, PDGF, and CS**F-1.**

Answer 52

Normal RAS proteins are tethered to the cytoplasmic aspect of the plasma membrane, as well as the endoplasmic reticulum and Golgi membranes. They can be activated by growth factor binding to receptors at the plasma membrane. [40] RAS is a member of a family of small G proteins that bind guanosine nucleotides (guanosine triphosphate, GTP and guanosine diphosphate, GDP), similar to the larger trimolecular G proteins. Normally RAS proteins flip back and forth between an excited signal-transmitting state and a quiescent state. In the inactive state, RAS proteins bind GDP. Stimulation of cells by growth factors leads to exchange of GDP for GTP and subsequent conformational changes that generates active RAS ( Fig. 7-26 ). The activated RAS stimulates downstream regulators of proliferation, such as the mitogen-activated protein (MAP) kinase cascade , which floods the nucleus with signals for cell proliferation.

Answer 53

When a normal cell is stimulated through a growth factor receptor, inactive (GDP-bound) RAS is activated to a GTP-bound state. Activated RAS recruits RAF and stimulates the MAP-kinase pathway to transmit growthpromoting signals to the nucleus. The mutated RAS protein is permanently activated because of inability to hydrolyze GTP, leading to continuous stimulation of cells without any external trigger. The anchoring of RAS to the cell membrane by the farnesyl moiety is essential for its action. See text for explanation of abbreviations.

Answer 54

(1) **nucleotide exchange** (GDP by GTP), which activates RAS protein, and (2) **GTP hydrolysis,** which converts the GTPbound, active RAS to the GDP-bound, inactive form. Both these processes are extrinsically regulated by other proteins.

Answer 55

the GTP-binding pocket or the enzymatic region essential for GTP hydrolysis, and thus markedly reduce the GTPase activity of the RAS protein. Mutated RAS is trapped in its activated GTP-bound form, and the cell is forced into a continuously proliferating state. It follows from this scenario that the consequences of mutations in RAS protein would be mimicked by mutations in the GAPs that fail to activate the GTPase activity and thus restrain normal RAS proteins. Indeed, disabling mutation of neurofibromin 1, a GAP, is associated with the inherited cancer syndrome familial neurofibromatosis type 1 ( Chapter 27 ).

Answer 56

BRAF This suggests that dysregulation of the RAS/RAF/MAP kinase pathway may be one of the initiating events in the development of melanomas, although it is not sufficient by itself to cause tumorigenesis. Indeed, BRAF mutations alone lead to oncogene-induced senescence giving rise to benign nevi rather than malignant melanoma. Thus, oncogene-induced senescence is a barrier to carcinogenesis that must be overcome by mutation and disabling of key protective mechanisms, such as those provided by the p53 gene (discussed later).

Answer 57

c-ABL tyrosine kinase

Answer 58

CML and some acute lymphoblastic leukemias,

Answer 59

the BCR moiety promotes the selfassociation of BCR-ABL. This is a common theme, since many different oncogenic tyrosine kinases consist of fusion proteins in which the non–tyrosine kinase partner drives selfassociation.

Answer 60

imatinib mesylate, a **“designer”** drug with **low toxicity and high therapeutic efficacy that inhibits the BCRABL** **kinase.** [47] [48] [49] This is another example of rational drug design emerging from an understanding of the molecular basis of cancer. It is also an example of the concept of oncogene addiction. Despite accumulation of numerous mutations throughout the genome, signaling through the BCR-ABL gene is required for the tumor to persist, hence inhibition of its activity is effective therapy.

Answer 61

**BCR-ABL** translocation The remaining mutations are selected for, and built around, the constant signaling through BCR-ABL. BCR-ABL signaling can be seen as the central lodgepole around which the structure is built. **Remove the lodgepole by inhibition of the BCR-ABL kinase, and the structure collapse**

Answer 62

**point mutations in the tyrosine kinase JAK2** The aberrant JAK2 kinase in turn activates transcription factors of the STAT family, **which promote the growth factor–independent proliferation and survival of the tumor cells.** Recognition of this molecular lesion has led to trials of JAK2 inhibitors in myeloproliferative disorders, and stimulated searches for activating mutations in other nonreceptor tyrosine kinases in a wide variety of human cancers.

Answer 63

Just as all roads lead to Rome, all signal transduction pathways converge to the nucleus, where a large bank of responder genes that orchestrate the cell's orderly advance through the mitotic cycle are activated. Indeed, the ultimate consequence of signaling through oncogenes like RAS or ABL is **inappropriate and continuous stimulation of nuclear transcription factors that drive growth-promoting genes.** ***Transcription factors contain specific amino acid** sequences or motifs that **allow them to bind DNA or to dimerize for DNA binding**.****Binding of these proteins to specific sequences in the genomic DNA activates transcription of genes.***

Answer 64

**Growth autonomy** may thus occur as a consequence of mutations affecting genes that regulate transcription.

Answer 65

MYC, MYB, JUN, FOS, and REL ``` Of these, MYC is most commonly involved in human tumors , and hence a brief overview of its function is warranted. ```

Answer 66

The MYC proto-oncogene is **expressed in virtually all eukaryotic cells and belongs to the immediate early response genes, which are rapidly induced when quiescent cells receive a signal to divide** (see discussion of liver regeneration in Chapter 3 ). After a transient increase of MYC messenger RNA, the expression declines to a basal level. ``` The **molecular basis of MYC function in cell replication is not entirely clear**. ``` As with many transcription factors, it is thought that MYC is involved in carcinogenesis by activating genes that are involved in proliferation

Answer 67

cell proliferation

Answer 68

* histone acetylation * , reduced cell adhesion, * increased cell motility * , increased telomerase activity, * increased protein synthesis, * decreased proteinase activity, * and other changes in cellular matbolism that enable a high rate of cell division.

Answer 69

***origins than needed for normal cell division, or bypass checkpoints involved in replication, leading to genomic damage and*** ***accumulation of mutations***. Finally, MYC is one of a handful of transcription factors that can act in concert to reprogram somatic cells into pluripotent stem cells ( Chapter 3 ); MYC may also enhance self-renewal, block differentiation, or both.

Answer 70

**Burkitt lymphoma, a B-cell tumor**

Answer 71

N-MYC and L-MYC

Answer 72

the **orderly progression** **of cells through the various phases of the cell cycle** is orchestrated by cyclin-dependent kinases (CDKs), which are activated by binding to cyclins, so called because of the **cyclic** **nature of their production and degradation.** **" SIGNAL LIGHT"**

Answer 73

cyclins D, E, A, and B "BEAD" DNAs are like BEADS

Answer 74

FIGURE 7-29 Schematic illustration of the role of cyclins, CDKs, and CDK inhibitors (CDKIs) in regulating the cell cycle. The shaded arrows represent the phases of the cell cycle during which specific cyclin-CDK complexes are active. As illustrated, cyclin D–CDK4, cyclin D –CDK6, and cyclin E–CDK2 regulate the G1-to-S transition by phosphorylation of the RB protein (pRB). Cyclin A–CDK2 and cyclin A–CDK1 are active in the S phase. Cyclin B–CDK1 is essential for the G2-to-M transition. Two families of CDKIs can block activity of CDKs and progression through the cell cycle. The so-called INK4 inhibitors, composed of p16, p15, p18, and p19, act on cyclin D–CDK4 and cyclin D–CDK6. The other family of three inhibitors, p21, p27, and p57, can inhibit all CDKs.

Answer 75

CDK4 CDK2 CDK1

Answer 76

Forms a complex with cyclin D that phosphorylates RB, **allowing the cell to progress through the G1 restriction point.**

Answer 77

Forms a complex with cyclin E in late G1, which is involved in G1/S transition. Forms a complex with cyclin A at the S phase that facilitates G2/M transition.

Answer 78

Forms a complex with cyclin B that facilitates G2/M transition.

Answer 79

* CIP/KIP family: p21, p27 (CDKN2A-C) * INK4/ARF family (CDKN1A-D)

Answer 80

Block the cell cycle by binding to cyclin-CDK complexes; p21 is induced by the tumor suppressor p53; p27 responds to growth suppressors such as TGF-β.

Answer 81

p16/INK4a binds to cyclin D–CDK4 and promotes the inhibitory effects of RB; p14/ARF increases p53 levels by inhibiting MDM2 activity.

Answer 82

p53 Ataxiatelangiectasia mutated

Answer 83

Tumor suppressor gene altered in the majority of cancers; **causes cell cycle arrest and apoptosis**. Acts mainly through p21 to cause cell cycle arrest. **Causes apoptosis** **by inducing the transcription of pro-apoptotic genes such as BAX .** p53 is required for the G1/S checkpoint and is a main component of the G2/M checkpoint.

Answer 84

**Activated by mechanisms that sense double-stranded DNA breaks.** **Transmits signals to arrest the cell cycle after DNA damage**. Acts through p53 in the G1/S checkpoint. At the G2/M checkpoint, it acts both through p53-dependent mechanisms and through the inactivation of CDC25 phosphatase, which disrupts the cyclin B–CDK1 complex. Component of a network of genes that include BRCA1 and BRCA2, which link DNA damage with cell cycle arrest and apoptosis.

Answer 85

affecting the breast, esophagus, liver, and a subset of lymphomas

Answer 86

melanomas, sarcomas, and glioblastomas.

Answer 87

* p21 (CDKN1A), * p27 (CDKN1B), and * p57 (CDKN1C)

Answer 88

* p15 (CDKN2B), * p16 (CDKN2A), * p18 (CDKN2C), and * p19 (CDKN2D)

Answer 89

**p16 (CDKN2A)**

Answer 90

* **75% of pancreatic carcinomas,** * **40% to 70% of glioblastomas,** * **50% of esophageal cancers,** * **20% to 70% of acute** **lymphoblastic leukemias,and** * **20% of non-small-cell lung carcinomas, soft-tissue sarcomas, and bladder cancers**

Answer 91

**internal controls of the cell cycle** called checkpoints,

Answer 92

* one at the **G1/S** transition * and the other at **G2/M.**

Answer 93

The **S phase** is the point of no return in the cell cycle.

Answer 94

Before a cell makes the final commitment to replicate, the **G1/S checkpoint** ***checks for DNA damage***; if damage is present, the DNA-repair machinery and mechanisms that arrest the cell cycle are put in motion. The delay in cell cycle progression provides the time needed for DNA repair; if the damage is not repairable, apoptotic pathways are activated to kill the cell. Thus, the **G1/S checkpoint prevents the replication of cells that have defects in DNA, which would be perpetuated as mutations or chromosomal breaks in the progeny of the cell.** DNA damaged after its replication can still be repaired as long as the chromatids have not separated.

Answer 95

The G2/M checkpoint **monitors the completion of DNA replication**and**checks whether the cell can safely initiate mitosis and separate sister chromatids**. This checkpoint is **particularly important in cells exposed to ionizing radiatio**n. Cells damaged by ionizing radiation activate the G2/M checkpoint and arrest in G2; defects in this checkpoint give rise to chromosomal abnormalities.

Answer 96

* as sensors, * proteins of the RAD family * and ataxia telangiectasia mutated (ATM) and as transducers, * the CHK kinase families. [59]

Answer 97

p53, which induces the cell cycle inhibitor p21.

Answer 98

both p53-dependent and p53-independent mechanisms

Answer 99

TUMOR SUPPRESSOR GENES

Answer 100

brakes to cell proliferation

Answer 101

RB and p53

Answer 102

* TGF-β receptor * E-cadherin * NF1 * NF2 * APC/β- catenin * PTEN * SMAD2 and SMAD4 * RB1 * p53 * WT1 * P16/INK4a * BRCA1 and BRCA2

Answer 103

* TGF-β receptor * E- cadherin

Answer 104

* Function : Growth inhibition * Tumors Associated : Carcinomas of colon with Somatic Mutations * Tumors Assocated: Unknown with Inherited Mutations

Answer 105

* Function : Cell adhesion * Tumors Associated:Carcinoma of stomach with Somatic Mutations * Familial gastric cancer

Answer 106

* Function: Inhibition of RAS signal transduction and of p21 cell cycle inhibitor * Tumors Associated :Neuroblastomas with Somatic Mutations * Tumors Assocated : Neurofibromatosis type 1 and sarcomas with Inherited Mutations

Answer 107

* Function : Cytoskeletal stability * Tumors Associated : Schwannomas and meningiomas with Somatic Mutations * Tumors Assocated: Neurofibromastosis type 2, acoustic schwannomas, and meningiomas with Inherited Mutations

Answer 108

* APC/β- catenin * PTEN * SMAD2 and SMAD4

Answer 109

* Function : **Inhibition of signal transduction** * Tumors Associated with Somatic Mutations: **Carcinomas of stomach, colon, pancreas; melanoma** * Tumors Assocated with Inherited Mutations : **Familial adenomatous polyposis coli/colon cancer**

Answer 110

* Function :PI3 kinase signal transduction * Tumors Associated with Somatic Mutations: * Endometrial and prostate cancers * Tumors Assocated with Inherited Mutations * Cowden syndrome

Answer 111

* Function :TGF-β signal transduction * Tumors Associated with Somatic Mutations: * Colon, pancreas tumors * Tumors Assocated with Inherited Mutations : Unknown

Answer 112

* RB1 * p53 * WT1 * P16/INK4a * BRCA1 and BRCA2

Answer 113

* Function: Regulation of cell cycle * Tumors Associated with Somatic Mutations * Retinoblastoma; osteosarcoma carcinomas of breast, colon, lung * Tumors Assocated with Inherited Mutations * Retinoblastomas, osteosarcoma

Answer 114

* Function : Cell cycle arrest and apoptosis in response to DNA damage * Tumors Associated with Somatic Mutations * Most human cancers * Tumors Assocated with Inherited Mutations * Li-Fraumeni syndrome; multiple carcinomas and sarcomas

Answer 115

* Function : Nuclear transcription * Tumors Associated with Somatic Mutations * Wilms' tumor * Tumors Assocated with Inherited Mutations * Wilms' tumor

Answer 116

* Function : Regulation of cell cycle by inhibition of cyclindependent kinases * Tumors Associated with Somatic Mutations: * Pancreatic, breast, and esophageal cancers * Tumors Assocated with Inherited Mutations * Malignant melanoma

Answer 117

* Function : DNA repair * Tumors Associated with Somatic Mutations: * Unknown * Tumors Assocated with Inherited Mutations * Carcinomas of female breast and ovary; carcinomas of male breast

Answer 118

RB protein, the **product of the RB gene**, is a **ubiquitously expressed nuclear phosphoprotein** that **plays a key role in regulating the cell cycle.**

Answer 119

* an active hypophosphorylated * state in quiescent cells and an * inactive hyperphosphorylated state * in the G1/S cell cycle transition

Answer 120

G1, or the gap between mitosis (M) and DNA replication (S).

Answer 121

* Gap 1 (G1) **between mitosis (M) and DNA** **replication (S),** and * Gap 2 (G2) between **DNA replication (S) and mitosis (M)** (see Fig. 7-29 ).

Answer 122

G1 to S Once cells cross the * *G1 checkpoint they can pause the cell cycle for a time,** but t**hey are obligated to complete mitosis. **

Answer 123

quiescence

Answer 124

senescence.

Answer 125

FIGURE 7-31 The role of RB in regulating the G1-S checkpoint of the cell cycle. Hypophosphorylated RB in complex with the E2F transcription factors binds to DNA, recruits chromatin-remodeling factors (histone deacetylases and histone methyltransferases), and inhibits transcription of genes whose products are required for the S phase of the cell cycle. When RB is phosphorylated by the cyclin D–CDK4, cyclin D–CDK6, and cyclin E–CDK2 complexes, it releases E2F. The latter then activates transcription of S-phase genes. The phosphorylation of RB is inhibited by CDKIs, because they inactivate cyclin-CDK complexes. Virtually all cancer cells show dysregulation of the G1-S checkpoint as a result of mutation in one of four genes that regulate the phosphorylation of RB; these genes are RB1, CDK4, the genes encoding cyclin D proteins, and CDKN2A (p16). EGF, epidermal growth factor; PDGF, platelet-derived growth factor; TGF-β, transforming growth factor-beta.

Answer 126

* cyclin E–CDK2 complexes, * and expression of cyclin E is dependent on the E2F family of transcription factors.

Answer 127

* First, it sequesters E2F, preventing it from interacting with other transcriptional activators. * Second, RB recruits chromatin-remodeling proteins, such as histone deacetylases and histone methyltransferases, which bind to the promoters of E2F-responsive genes such as cyclin E. These enzymes modify chromatin so as to **make promoters insensitive to transcription factors.**

Answer 128

Mitogenic signaling leads to cyclin D expression and activation of cyclin D–CDK4/6 complexes. * *These complexes phosphorylate RB,** * *inactivating the protein** and **releasing E2F to induce target genes such as cyclin E.**

Answer 129

Expression of cyclin E then **stimulates DNA replication and progression through the cell cycle**.

Answer 130

When the cells enter S phase, **they are committed to divide without additional growth factor stimulation.** During the ensuing M phase the phosphate groups are removed from RB by cellular phosphatases, regenerating the hypophosphorylated form of RB. E2Fs are not the sole effectors of Rb-mediated G1 arrest. Rb also controls the stability of the cell cycle inhibitor p27

Answer 131

the **molecular brakes on the cell cycle are released,** and the **cells move through the** **cell cycle.**

Answer 132

The mutations of RB genes found in tumors are localized to a region of the RB protein, called the “RB pocket,” that is involved in **binding to E2F**. However, the versatile RB protein has also been shown to bind to a variety of other transcription factors that regulate cell differentiation. [65] For example, RB stimulates myocyte-, adipocyte-, melanocyte-, and macrophage-specific transcription factors. Thus, the RB pathway couples control of cell cycle progression at G1 with differentiation, which may explain how differentiation is associated with exit from the cell cycle. In addition to these dual activities, RB can also induce senescence, discussed below

Answer 133

The reason for the occurrence of tumors restricted to the retina in persons who inherit one defective allele of **RB is not fully understood,** but some possible explanations have emerged from the study of mice with targeted disruption of the rb locus. For instance, RB family members may partially complement its function in cell types other than retinoblasts. Indeed, RB is a member of a small family of proteins, so-called pocket proteins, which also include **p107 and p130.** [66] **All three proteins bind to E2F transcription** factors. The complexity grows; there are seven **E2F proteins (named E2F1 through E2F7),** which **function as either transcriptional activators** or repressors. The pocket proteins are all thought to regulate progression through the cell cycle as well as differentiation in a manner similar to that described for RB above. However, each member of this protein family binds a different set of E2F proteins and is also expressed at different times in the cell cycle. Thus, although there is some redundancy in the network, their functions are not completely overlapping. The complexity of the pocket protein–E2F network is just now being unraveled. For example, in a mouse model of retinoblastoma, it has been shown that mutation of different members of the network in various combinations generates retinoblastomas not just from retinoblasts, but also from differentiated cells in the retina, such as horizontal interneurons

Answer 134

With respect to the second question (i.e., why the loss of RB is not more common in human tumors), the answer is much simpler: **Mutations in other genes that control RB phosphorylation can mimic the effect of RB loss**, and such genes are **mutated in many cancers that may have normal RB genes.** Thus, for example, **mutational activation of cyclin D or CDK4 would favor cell proliferation by facilitating RB phosphorylation**. As previously discussed, **cyclin D is overexpressed**in many tumors**because of gene amplification or translocation.** Mutational inactivation of CDKIs would also drive the cell cycle by unregulated activation of cyclins and CDKs. Thus, the emerging paradigm is that loss of normal cell cycle control is central to malignant transformation and that at least one of four key regulators of the cell cycle ( p16/INK4a, cyclin D, CDK4, RB) is dysregulated in the vast majority of human cancers . [68] In cells that harbor mutations in any one of these other genes, the function of RB is disrupted even if the RB gene itself is not mutated

Answer 135

by neutralizing the growth-inhibitory activities of RB. In these cases, RB protein is functionally inactivated by the binding of a viral protein and no longer acts as a cell cycle inhibitor. Simian virus 40 and polyomavirus large T antigens, adenoviruses EIA protein, and HPV E7 protein all bind to the hypophosphorylated form of RB. The binding occurs in the same RB pocket that normally sequesters E2F transcription factors; in the case of HPV the binding is particularly strong for viral types, such as HPV type 16, that confer high risk for the development of cervical carcinomas. Thus, the RB protein, unable to bind the E2F transcription factors, is functionally inactivated, and the transcription factors are free to cause cell cycle progression.

Answer 136

p53: Guardian of the Genome.

Answer 137

The p53 gene is located on **chromosome 17p13.1,** and it is the **most common target for genetic alteration in human tumors** **(The official name of the gene is TP53 and the protein is p53; for the sake of simplicity, we refer to both as “p53”.) A little over 50% of human tumors contain mutations in this gene. Homozygous loss of p53 occurs in virtually every type of cancer, including carcinomas of the lung, colon, and breast—the three leading causes of cancer death.**

Answer 138

Li-Fraumeni syndrome

Answer 139

sarcomas, breast cancer, leukemia, brain tumors, and carcinomas of the adrenal cortex.

Answer 140

The fact that p53 mutations are common in a variety of human tumors suggests that the p53 protein functions as a **critical gatekeeper against the formation of cancer**. Indeed, it is evident that **p53 acts as a “molecular policeman**” that prevents the propagation of genetically damaged cells. p53 is a **transcription factor that is at the center of a large network of signals that sense cellular stress, such as DNA damag**e,**shortened telomeres, and hypoxia.** Many activities of the p53 protein are **related to its function as a transcription factor.** Several hundred genes have been shown to be regulated by p53 in numerous different contexts, but which genes are the key for the p53 response is not yet clear. Approximately 80% of the p53 point mutations present inhuman cancers are located in the **DNA-binding domain of the protein**. However, the effects of different point mutations vary considerably; in some cases there is complete abrogation of transcriptional capabilities, whereas other mutants retain the ability to bind to and activate a subset of genes. In addition to somatic and inherited mutations, p53 functions can be inactivated by other mechanisms. As with RB, the transforming proteins of several DNA viruses, including the E6 protein of HPV, can bind to and promote the degradation of p53. Also, analagous to RB, it is thought that in the majority of tumors without a p53 mutation, the function of the p53 pathway is blocked by mutation in another gene that regulates p53 function. For example, MDM2 and MDMX stimulate the degradation of p53; these proteins are frequently overexpressed in malignancies in which the gene encoding p53 is not mutated. Indeed, MDM2 is amplified in 33% of human sarcomas, thereby causing functional loss of p53 in these tumors

Answer 141

* activation of temporary cell cycle arrest **(quiescence),** * induction of permanent cell cycle arrest (senescence), * or triggering of programmed cell death **(apoptosis).**

Answer 142

because of its association **with MDM2**, a protein tha**t targets it for destruction.**

Answer 143

p53 undergoes **post-transcriptional modifications** that **release it from MDM2** and increase its half-life. Unshackled from MDM2, p53 also becomes activated as a transcription factor.

Answer 144

* those that cause cell cycle arrest and those that cause apoptosis. If DNA damage can be repaired during cell cycle arrest, the cell reverts to a normal state; * if the repair fails, p53 induces apoptosis or senescence. Recently, however, the plot has thickened. It has been known that repression of a subset of proproliferative and anti-apoptotic genes is key to the p53 response, but it was not clear how p53 achieved repression, since in most assays it seemed to be an activator of transcription. At this point enter the recently famous miRNAs, the small guys with big clubs. It has been shown that p**53 activates transcription of the mir34 family of miRNAs (mir34a–mir34c). [**76] miRNAs, as discussed in Chapter 5 , **bind to cognate sequences in the 3′ untranslated region of mRNAs,** **preventing translation** ( Fig. 7-32B ). Interestingly, blocking mir34 severely hampered the p53 response in cells, while ectopic expression of mir34 without p53 activation is sufficient to induce growth arrest and apoptosis. Thus, mir34 microRNAs are able to recapitulate many of the functions of p53 and are necessary for these functions, demonstrating the importance of mir34 to the p53 response. Targets of mir34s include pro-proliferative genes such as cyclins, and anti-apoptotic genes such as BCL2. p53 regulation of mir34 explains, at least in part, how p53 is able to repress gene expression, and it seems that regulation of this miRNA is crucial for the p53 response.

Answer 145

FIGURE 7-32 A, The role of p53 in maintaining the integrity of the genome. Activation of normal p53 by DNA-damaging agents or by hypoxia leads to cell cycle arrest in G1 and induction of DNA repair, by transcriptional up-regulation of the cyclin-dependent kinase inhibitor CDKN1A (p21) and the GADD45 genes. Successful repair of DNA allows cells to proceed with the cell cycle; if DNA repair fails, p53 triggers either apoptosis or senescence. In cells with loss or mutations of p53, DNA damage does not induce cell cycle arrest or DNA repair, and genetically damaged cells proliferate, giving rise eventually to malignant neoplasms. B, p53 mediates gene repression by activating transcription of miRNAs. p53 activates transcription of the mir34 family of miRNAs. mir34s repress translation of both proliferative genes, such as cyclins, and anti-apoptotic genes, such as BCL2. Repression of these genes can promote either quiescence or senescence as well as apoptosis.

Answer 146

* ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia and * Rad3 related (ATR). [77,] [78]

Answer 147

As the name implies, the ATM gene was originally identified as the **germ-line mutation** in **individuals with ataxia-telangiectasia**. Persons with this disease, which is characterized by an inability to repair certain kinds of DNA damage, suffer from an increased incidence of cancer. The types of damage sensed by ATM and ATR are different, but the downstream pathways they activate are similar. Once triggered, both ATM and ATR phosphorylate a variety of targets, including p53 and DNA-repair proteins. Phosphorylation of these two targets leads to a pause in the cell cycle and stimulation of DNA-repair pathways, respectively.

Answer 148

by specific changes in morphology and gene expression that differentiate it from quiescence or reversible cell cycle arrest.

Answer 149

Senescence requires **activation of p53 and/or RB** and **expression of their mediators,** * *such as the CDK inhibitors,** and is generally **irreversible, although it may require the continued** * *expression of p53.** The mechanisms of senescence are unclear but involve epigenetic changes that result in the formation of heterochromatin at different loci throughout the genome. These senescence-associated heterochromatin foci include pro-proliferative genes regulated by E2F; this drastically and permanently alters expression of these E2F targets. Like all p53 responses, senescence may be stimulated in response to a variety of stresses, such as unopposed oncogene signaling, hypoxia, and shortened telomeres.

Answer 150

p53-induced apoptosis of cells with irreversible DNA damage is the ultimate protective mechanism against neoplastic transformation. p53 directs the transcription of several proapoptotic genes such as **BAX and PUMA** (approved name BBC3; described later). Exactly how a cell decides whether to repair its DNA or to enter apoptosis is unclear. It appears that the * *affinity of p53 for the promoters and enhancers of DNA-repair genes is stronger than its affinity** * *for pro-apoptotic genes.** [80] Thus, the DNA-repair pathway is stimulated first, while p53 continues to accumulate. Eventually, if the DNA damage is not repaired, enough p53 accumulates to stimulate transcription of the pro-apoptotic genes and the cell dies. While this scheme is generally correct, there seem to be important cell type–specific responses as well, with some cell types succumbing to apoptosis early, while others opt for senescence. [80] Such differential responses may be related to the functions of other p53 family members expressed in different cell types (see below).

Answer 151

``` Adenomatous polyposis coli genes (APC) represents a **class of tumor suppressors whose main function is to down-regulate growth-promoting signals.** ```

Answer 152

APC is a **component of the WNT signaling pathway**, which has a **major role in controlling cell** **fate, adhesion, and cell polarity** during embryonic development ( Fig. 7-33 ). WNT signaling is **also required for self-renewal of hematopoietic stem cells**. WNT signals through a family of cell surface receptors called **frizzled (FRZ)**, and stimulates several pathways, the central one involving β-catenin and APC.

Answer 153

FIGURE 7-33 A, The role of APC in regulating the stability and function of β-catenin. APC and β-catenin are components of the WNT signaling pathway. In resting cells (not exposed to WNT), β-catenin forms a macromolecular complex containing the APC protein. This complex leads to the destruction of β-catenin, and intracellular levels of β-catenin are low. B, When cells are stimulated by WNT molecules, the destruction complex is deactivated, β-catenin degradation does not occur, and cytoplasmic levels increase. β-catenin translocates to the nucleus, where it binds to TCF, a transcription factor that activates genes involved in cell cycle progression. C, When APC is mutated or absent, the destruction of β-catenin cannot occur. β-catenin translocates to the nucleus and coactivates genes that promote entry into the cell cycle, and cells behave as if they are under constant stimulation by the WNT pathway.

Answer 154

In the absence of WNT signaling **APC causes degradation of β-catenin, preventing its accumulation in the cytoplasm**. [85] It does so by forming a **macromolecular complex with β-catenin, axin, and GSK3β**, which**leads to the phosphorylation and eventually ubiquitination of β-catenin and destruction by the proteasome.** Signaling by WNT blocks the APC-AXIN-GSK3β destruction complex, allowing β-catenin to translocate from the cytoplasm to the nucleus. In the cell nucleus, **β-catenin forms a complex with TCF**, a transcription factor that up-regulates cellular proliferation by i**ncreasing the transcription of c-MYC, cyclin D1**, and other genes. Since inactivation of the APC gene disrupts the destruction complex, β-catenin survives and translocates to the nucleus, where it can activate transcription in cooperation with TCF. [85] Thus, cells with loss of APC behave as if they are under continuous WNT signaling. The importance of the APC/β-catenin signaling pathway in tumorigenesis is attested to by the fact that colon tumors that have normal APC genes harbor mutations in β-catenin that prevent its destruction by APC, allowing the mutant protein to accumulate in the nucleus. Dysregulation of the APC/β-catenin pathway is not restricted to colon cancers; mutations in the β-catenin gene are present in more than 50% of hepatoblastomas and in approximately 20% of hepatocellular carcinomas.

Answer 155

β-catenin binds to the cytoplasmic tail of Ecadherin, **a cell surface protein that maintains intercellular adhesiveness** . Loss of cell-cell contact, such as in a wound or injury to the epithelium, **disrupts the interaction between Ecadherin** **and β-catenin**, and **allows β-catenin to travel to the nucleus and stimulate proliferation;** this is an **appropriate response to injury that can help repair the wound**. Re-establishment of these **E-cadherin contact**s as the wound heals leads to β-catenin again being sequestered at the membrane and reduction in the proliferative signal; these cells are said to be “contactinhibited.” Loss of contact inhibition, by mutation of the E-cadherin/β-catenin axis, or by other methods, is a key characteristic of carcinomas. Furthermore, loss of cadherins can favor the malignant phenotype by allowing easy disaggregation of cells, which can then invade locally or metastasize. Reduced cell surface expression of E-cadherin has been noted in many types of cancers, including those that arise in the esophagus, colon, breast, ovary, and prostate. [87] Germline mutations of the E-cadherin gene can predispose to familial gastric carcinoma, and mutation of the gene and decreased E-cadherin expression are present in a variable proportion of gastric cancers of the diffuse type. The molecular basis of reduced E-cadherin expression is varied. In a small proportion of cases, there are mutations in the E-cadherin gene (located on 16q); in other cancers, E-cadherin expression is reduced as a secondary effect of mutations in β-catenin genes. Additionally, E-cadherin may be down-regulated by transcription repressors, such as SNAIL, which have been implicated in epithelial-to-mesenchymal transition and metastasis

Answer 156

Also called the **CDKN2A gene locus**, the INK4a/ARF locus encodes two protein products; the p16/INK4a CDKI, **which blocks cyclin D/CDK2**-**mediated phosphorylation of RB, keeping the RB checkpoint in place**. The second gene product, p14/ARF, activates the p53 pathway by inhibiting MDM2 and preventing destruction of p53 . Both protein products function as tumor suppressors, and thus mutation or silencing of this locus impacts both the RB and p53 pathways. p16 in particular is crucial for the induction of senescence. Mutations at this locus have been detected in bladder, head and neck tumors, acute lymphoblastic leukemias, and cholangiocarcinomas. In some tumors, such as cervical cancer, p16/INK4a is frequently silenced by hypermethylation of the gene, without the presence of a mutation (see discussion of epigenetic changes). The other CDKIs also function as tumor suppressors and are frequently mutated or otherwise silenced in many human malignancies, including 20% of familial melanomas, 50% of sporadic pancreatic adenocarcinomas, and squamous cell carcinomas of the esophagus.

Answer 157

In most normal epithelial, endothelial, and hematopoietic cells, **TGF-β is a potent inhibitor** of **proliferation**. It **regulates cellular processes** by **binding to a serine-threonine kinase complex** composed of TGF-β receptors I and II. Dimerization of the receptor upon ligand binding leads to activation of the kinase and phosphorylation of receptor SMADs (R-SMADs). Upon phosphorylation, R-SMADs can enter the nucleus, bind to SMAD-4, and activate transcription of genes, including the CDKIs p21 and p15/INK4b. In addition, TGF-β signaling leads to repression of c-MYC, CDK2, CDK4, and cyclins A and E. As can be inferred from our earlier discussion, these changes result in decreased phosphorylation of RB and cell cycle arrest.

Answer 158

These **mutations may affect the type II TGF-β** **receptor or interfere with SMAD** molecules that serve to transduce antiproliferative signals from the receptor to the nucleus.

Answer 159

colon, stomach, and endometrium.

Answer 160

pancreatic cancers. In 100% of pancreatic cancers and 83% of colon cancers, at least one component of the TGF-β pathway is mutated . However, in many cancers, loss of TGF-β-mediated growth inhibition occurs at a level downstream of the core signaling pathway, for example, loss of p21 and/or persistent expression of c-Myc. These tumor cells can then use other elements of the TGFβ–induced program, **including immune system suppression/evasion or promotion of** **angiogenesis, to facilitate tumor progression**. [89] Thus TGF-β can function to prevent or promote tumor growth, depending on the state of other genes in the cell.

Answer 161

PTEN (Phosphatase and tensin homologue) is a **membrane-associated phosphatase encoded by a gene on chromosome 10q23** that is mutated in

Answer 162

Cowden syndrome, an **autosomal dominant** **disorder marked by frequent benign growths,** such as **tumors of the skin appendages, and an increased incidence of epithelial cancers, particularly of the breast**( Chapter 23 ),**endometrium, and thyroid.**

Answer 163

PTEN acts as a tumor suppressor by serving as a brake on the prosurvival/ pro-growth PI3K/AKT pathway

Answer 164

As you will recall from Chapter 3 , this pathway is **normally stimulate**d (along with the RAS and JAK/STAT pathways) when ligands bind to **receptor tyrosine kinases and involves a cascade of phosphorylation events.** First, PI3K (phosphoinositide 3-kinase) phosphorylates the lipid inositide-3-phosphate to give rise to inositide-3,4,5-triphosphate, which binds and activates the kinase PDK1. PDK1 and other factors in turn phosphorylate and activate the serine/threonine kinase AKT, which is a major node in the pathway with several important functions. By phosphorylating a number of substrates, including **BAD and MDM2**, AKT enhances cell survival.

Answer 165

TSC1 and TSC2 are the products of two tumor suppressor genes that are mutated in **tuberous sclerosi**s ( Chapter 28 ), an autosomal dominant disorder associated with developmental malformations and unusual benign neoplasms such as cardiac rhabdomyomas ( Chapter 12 ), renal angiomyolipomas, and giant cell astrocytomas.

Answer 166

``` tuberous sclerosis ( Chapter 28 ), an autosomal dominant disorder associated with developmental malformations and unusual benign neoplasms such as cardiac rhabdomyomas ( Chapter 12 ), renal angiomyolipomas, and giant cell astrocytomas ```

Answer 167

Although acquired loss of PTEN function is one of the **most common ways that PI3K/AKT signaling** is upregulated in various cancers, many other components of the pathway, **including PI3K itself, may also be mutated so as to increase signaling.** Considering all of these molecular lesions collectively, it is said that this may be the **most commonly mutated pathway in human cancer**. As a result there is great interest in targeting the **PI3K/AKT pathway with inhibitors of mTOR, AKT, and other kinases in the pathway.**

Answer 168

Individuals who inherit one mutant allele of the NF1 gene develop **numerous benign** * *neurofibromas** and **optic nerve glioma**s as a result of inactivation of the second copy of the gene. [92] This condition is called **neurofibromatosis type 1** ( Chapter 27 ). Some of the neurofibromas later develop into malignant peripheral nerve sheath tumors. Neurofibromin, the protein product of the NF1 gene, contains a GTPase-activating domain, which regulates signal transduction through RAS proteins. Recall that RAS transmits growth-promoting signals and flips back and forth between GDP-binding (inactive) and GTP-binding (active) states. **Neurofibromin facilitates conversion of RAS from an active to an inactive state.** With loss of neurofibromin function, RAS is trapped in an active, signal-emitting state.

Answer 169

Germline mutations in the NF2 gene predispose to the development of neurofibromatosis type 2. [93] As discussed in Chapter 27 **, individuals with mutations in NF2 develop benign bilateral schwannomas of the acoustic nerve**. In addition, somatic mutations affecting both alleles of NF2 have also been found in sporadic meningiomas and ependymomas.

Answer 170

The product of the NF2 gene, called **neurofibromin 2 or merlin**, shows a great **deal of homology with the red cell membrane cytoskeletal protein**4.1 ( Chapter 14 ), and is**related to the ERM (ezrin, radixin, and moesin)** family of membrane cytoskeleton-associated proteins. Although the mechanism by which merlin deficiency leads to carcinogenesis is not known, cells lacking this protein are not capable of establishing stable cell-to-cell junctions and are insensitive to normal growth arrest signals generated by cell-to-cell contact. Merlin is a key member of the Salvador- Warts-Hippo (SWH) tumor suppressor pathway, originally described in Drosophila. The signaling pathway controls organ size by modulating cell growth, proliferation, and apoptosis. Many human homologues of genes in the SWH pathway have been implicated in human cancers.

Answer 171

Germline mutations of the **von Hippel-Lindau (VHL)** gene on chromosome 3p are associated with hereditary renal cell cancers, pheochromocytomas, hemangioblastomas of the central nervous system, retinal angiomas, and renal cysts. [60] Mutations of the VHL gene have also been noted in **sporadic renal cell cancers** ( Chapter 20 ). The VHL protein is part of a ubiquitin ligase complex. **A critical substrate for this activity is HIF1α (hypoxia-inducible transcription factor 1α)**. In the presence of oxygen, **HIF1α** is **hydroxylated and binds to the VHL protein,** leading to **ubiquitination and proteasomal degradation**. This hydroxylation reaction requires oxygen; in hypoxic environments the reaction cannot occur, and HIF1α escapes recognition by VHL and subsequent degradation. HIF1α can then translocate to the nucleus and turn on many genes, such as the growth/angiogenic factors vascular endothelial growth factor (VEGF) and PDGF. Lack of VHL activity prevents ubiquitination and degradation of HIF1α and is associated with increased levels of angiogenic growth factors.

Answer 172

The WT1 gene, located on chromosome 11p13, is associated with the development of Wilms' tumor, a pediatric kidney cancer. [95] Both inherited and sporadic forms of Wilms' tumor occur, and mutational inactivation of the WT1 locus has been seen in both forms. The WT1 protein is a **transcriptional activator of genes involved in renal and gonadal differentiation**. **It regulates the mesenchymal-to-epithelial transition****that occurs in kidney development.** Though not precisely known, it is likely that the **tumorigenic effect of WT1 deficiency is intimately connected with the role of the gene in the differentiation of genitourinary tissues**. Interestingly, although WT1 is a tumor suppressor in Wilms' tumor, a variety of adult c**ancers, including leukemias and breast carcinomas**, have also been shown to overexpress WT1. Since these tissues do not normally express WT1 at all, it has been suggested that WT1 may function as an oncogene in these cancers. ``` Another Wilms' gene, WT2, located on 11p15, is associated with the Beckwith- Wiedemann syndrome ( Chapter 10 ). ```

Answer 173

PTCH1 and PTCH2 are tumor suppressor genes that **encode a cell membrane protein (PATCHED),**which functions as a**receptor for a family of proteins called Hedgehog**. [96] The Hedgehog/PATCHED pathway **regulates several genes, including TGF-β and PDGFRA** and **PDGFRB**.

Answer 174

Mutations in PTCH are related to Gorlin syndrome, an **inherited condition also known** **as nevoid basal cell carcinoma syndrome** (see Chapter 26 ). PTCH mutations are present in **20% to 50% of sporadic cases of basal cell carcinoma.** About one half of such mutations are of the type caused by UV exposure.

Answer 175

* the extrinsic and * intrinsic pathways.

Answer 176

* **death receptor CD95/Fas (extrinsic pathway)** and by * **DNAdamage (intrinsic pathway).**

Answer 177

The extrinsic pathway is initiated when **CD95/Fas binds to its ligand, CD95L/FasL**, leading to trimerization of the receptor and its cytoplasmic death domains, which **attract the intracellular adaptor protein FADD.** This protein **recruits procaspase 8 to form the death-inducing signaling complex.** Procaspase 8 is activated by cleavage into smaller subunits, generating caspase 8. Caspase 8 then activates downstream caspases such as **caspase 3**, a **typical executioner caspase that cleaves DNA and other substrates to cause cell death.**Additionally, caspase 8 can cleave and**activate the BH3-only protein BID,****activating the intrinsic pathway as well.**

Answer 178

The intrinsic pathway of apoptosis is triggered by a variety of stimuli, including withdrawal of survival factors, stress, and injury. Activation of this pathway leads to permeabilization of the mitochondrial outer membrane, with resultant release of molecules, such as **cytochrome c**, that **initiate apoptosis.**

Answer 179

BCL2 family of proteins

Answer 180

BAX and BAK

Answer 181

BCL2 and BCL-XL.

Answer 182

BAD, BID, and PUMA The BH3-only proteins sense death-inducing stimuli and promote apoptosis by neutralizing the actions of anti-apoptotic proteins like BCL2 and BCL-XL. When the sum total of all BH3 proteins expressed “overwhelms” the anti-apoptotic BCL2/BCL-XL protein barrier, BAX and BAK are activated and form pores in the mitochondrial membrane. Cytochrome c leaks into the cytosol, where it binds to APAF1, activating caspase 9. Like caspase 8 of the extrinsic pathway, caspase 9 can cleave and activate the executioner caspases. The caspases can be inhibited by a family of proteins called Inhibitors of Apoptosis Proteins (IAPs). Some tumors avoid apoptosis by upregulating these proteins, and there is interest in developing drugs that can block the interaction between IAPs and caspases. Because of the pro-apoptotic effect of BH3-only proteins, efforts are underway to develop BH3 mimetic drugs

Answer 183

FIGURE 7-34 CD95 receptor–induced and DNA damage–triggered pathways of apoptosis and mechanisms used by tumor cells to evade cell death. (1) Reduced CD95 level. (2) Inactivation of death-induced signaling complex by FLICE protein (caspase 8; apoptosis- related cysteine peptidase). (3) Reduced egress of cytochrome c from mitochondrion as a result of up-regulation of BCL2. (4) Reduced levels of pro-apoptotic BAX resulting from loss of p53. (5) Loss of apoptotic peptidase activating factor 1 (APAF1). (6) Up-regulation of inhibitors of apoptosis (IAP). FADD, Fas-associated via death domain.

Answer 184

TELOMERASE

Answer 185

As was discussed in the section on cellular aging ( Chapter 1 ), most normal human cells have a capacity of 60 to 70 doublings. After this, the cells lose their ability to divide and become senescent. This phenomenon has been ascribed to progressive shortening of telomeres at the ends of chromosomes

Answer 186

p53 and RB. In cells in which the checkpoints are disabled by p53 or RB1 mutations, the nonhomologous end-joining pathway is activated as a last-ditch effort to save the cell, joining the shortened ends of two chromosomes. [103] This inappropriately activated repair system results in dicentric chromosomes that are pulled apart at anaphase, resulting in new doublestranded DNA breaks. The resulting genomic instability from the repeated bridge-fusionbreakage cycles eventually produces mitotic catastrophe, characterized by massive cell death. It follows that for tumors to grow indefinitely, as they often do, loss of growth restraints is not enough. Tumor cells must also develop ways to avoid both cellular senescence and mitotic catastrophe ( Fig. 7-35 )

Answer 187

FIGURE 7-35 Sequence of events in the development of limitless replicative potential. Replication of somatic cells, which do not express telomerase, leads to shortened telomeres. In the presence of competent checkpoints, cells undergo arrest and enter nonreplicative senescence. In the absence of checkpoints, DNA-repair pathways are inappropriately activated, leading to the formation of dicentric chromosomes. At mitosis the dicentric chromosomes are pulled apart, generating random double-stranded breaks, which then activate DNA-repair pathways, leading to the random association of double-stranded ends and the formation, again, of dicentric chromosomes. Cells undergo numerous rounds of this bridge-fusion-breakage cycle, which generates massive chromosomal instability and numerous mutations. If cells fail to re-express telomerase, they eventually undergo mitotic catastrophe and death. Re-expression of telomerase allows the cells to escape the bridgefusion- breakage cycle, thus promoting their survival and tumorigenesis.

Answer 188

vascularized Like normal tissues, tumors require delivery of oxygen and nutrients and removal of waste products; presumably the **1- to 2-mm zone represents the maximal distance across which oxygen**, nutrients, and waste can diffuse from blood vessels.

Answer 189

The **vessels are leaky and dilated,** and have a **haphazard pattern of connection.**

Answer 190

* perfusion supplies needed nutrients and oxygen, * and newly formed endothelial cells stimulate the growth of adjacent tumor cells by secreting growth factors, such as insulin-like growth factors (IGFs), PDGF, and granulocytemacrophage colony-stimulating factor.

Answer 191

The emerging paradigm is that tumor angiogenesis is controlled by the **balance between angiogenesis promoters and inhibitors.** Early in their growth, most human tumors do not induce angiogenesis. They remain small or in situ, possibly for years, **until the angiogenic switch terminates this stage of vascular** **quiescence**. [105] The molecular basis of the angiogenic switch involves increased production of angiogenic factors and/or loss of angiogenic inhibitors.

Answer 192

Many proteases can release the **proangiogenic basic fibroblast growth factors (bFGF) stored in the ECM;** conversely, **three potent angiogenesis inhibitors—angiostatin, endostatin, and vasculostatin**—are produced by **proteolytic cleavage of plasminogen, collagen, and transthyretin, respectively**. The angiogenic switch is controlled by **several physiologic stimuli, such as hypoxia.** Relative lack of oxygen stimulates HIF1α, an oxygen-sensitive transcription factor mentioned above, which then **activates transcription of a variety of pro-angiogenic cytokines**, such as **VEGF and bFGF.** These factors create an angiogenic gradient that **stimulates the proliferation of endothelial cells** and guides the growth of new vessels toward the tumor.

Answer 193

VEGF also increases the expression of ligands that activate the Notch signaling pathway, which **plays a crucial role in regulating the** **branching and density of the new vessels (** Chapter 3 ). Both pro- and anti-angiogenic factors are regulated by many other genes frequently mutated in cancer. For example, in normal cells,

Answer 194

**Invasion and metastasis** are biologic hallmarks of malignant tumors. They are the major cause of cancer-related morbidity and mortality and hence are the subjects of intense scrutiny. Studies in mice and humans reveal that although millions of cells are released into the circulation each day from a primary tumor, only a few metastases are produced. Indeed, tumor cells can be frequently detected in the blood and marrow of patients with breast cancer who have not, and do not ever, develop gross metastatic disease.

Answer 195

Each step in the process is subject to a multitude of controls; hence, at any point in the sequence the breakaway cell may not survive. [109] For tumor cells to break loose from a primary mass, enter blood vessels or lymphatics, and produce a secondary growth at a distant site, they must go through a series of steps (summarized in Fig. 7-36 ).

Answer 196

* (1) invasion of the extracellular matrix (ECM); * (2) vascular dissemination, homing of tumor cells, and colonization. Subsequently, the molecular genetics of the metastatic cascade, as currently understood, will be presented

Answer 197

FIGURE 7-36 The metastatic cascade. Sequential steps involved in the hematogenous spread of a tumor.

Answer 198

basement membrane and interstitial connective tissue. Though organized differently, each of these components of ECM is made up of collagens, glycoproteins, and proteoglycans. As shown in Figure 7-36 , tumor cells must interact with the ECM at several stages in the metastatic cascade.

Answer 199

* **breach the underlying basement membran**e, * then traverse the interstitial connective tissue, and * ultimately gain access to the circulation by penetrating the vascular basement membrane. This process is repeated in reverse when tumor cell emboli extravasate at a distant site.

Answer 200

* Changes (“loosening up”) of tumor cell-cell interactions * Degradation of ECM * Attachment to novel ECM components * Migration of tumor cells

Answer 201

FIGURE 7-37 A–D, Sequence of events in the invasion of epithelial basement membranes by tumor cells. Tumor cells detach from each other because of reduced adhesiveness, then secrete proteolytic enzymes, degrading the basement membrane. Binding to proteolytically generated binding sites and tumor cell migration follow.

Answer 202

intercellular adhesion molecules. Normal cells are neatly glued to each other and their surroundings by a variety of adhesion molecules. [111]

Answer 203

Cell-cell interactions are mediated by the cadherin family of transmembrane glycoproteins. E-cadherins mediate homotypic adhesions in epithelial tissue, thus serving to keep the epithelial cells together and to relay signals between the cells; intracellularly the E-cadherins are connected to β-catenin and the actin cytoskeleton. In several epithelial tumors, including a**denocarcinomas of the colon and breast**, there is a **downregulation** **of E-cadherin expression.** Presumably, this down-regulation reduces the ability of cells to adhere to each other and facilitates their detachment from the primary tumor and their advance into the surrounding tissues. E-cadherins are linked to the cytoskeleton by the catenins, proteins that lie under the plasma membrane (see Fig. 7-33 ).

Answer 204

catenins. In some tumors E-cadherin is normal, but its expression is reduced because of mutations in the gene for α catenin

Answer 205

local degradation of the basement membrane and interstitial connective tissue

Answer 206

Tumor cells may either **secrete proteolytic enzymes** themselves o**r induce** **stromal cells (e.g., fibroblasts and inflammatory cells)** to elaborate proteases.

Answer 207

* matrix metalloproteinases (MMPs), * cathepsin D, * and urokinase plasminogen activator

Answer 208

MMPs regulate tumor invasion **not only by remodeling insoluble components** of the basement membrane and interstitial matrix **but also by releasing ECM-sequestered growth factors.** Indeed, cleavage products of collagen and proteoglycans also have chemotactic, angiogenic, and growthpromoting effects. [112] For example, **MMP9 is a gelatinase** that cleaves type IV collagen of the epithelial and vascular basement membrane and also stimulates release of VEGF from ECMsequestered pools. Benign tumors of the breast, colon, and stomach show little type IV collagenase activity, whereas their malignant counterparts overexpress this enzyme. Concurrently, the **concentrations of metalloproteinase inhibitors are reduced** so that the **balance is tilted greatly toward tissue degradation.** Indeed, overexpression of MMPs and other proteases has been reported for many tumors.

Answer 209

ameboid migration. [113] In this type of migration **the cell squeezes through spaces in the matrix instead** **of cutting its way through it.** This ameboid migration is **much quicker, and tumor cells seem to be** **able to use collagen fibers as high-speed railways in their travels.** Tumor cells, in vitro at least, seem to be able to switch between the two forms of migration, perhaps explaining the disappointing performance of MMP inhibitors in clinical trials.

Answer 210

attachment of tumor cells to ECM proteins . Normal epithelial cells have receptors, such as **integrins**, for **basement membrane laminin and collagens that are polarized at their basal surface;**these receptors**help to maintain the cells in a resting, differentiated state.** Loss of adhesion in normal cells leads to induction of apoptosis, while, not surprisingly, **tumor cells are resistant to this form of cell death**. Additionally, the matrix **itself is modified in ways that promote invasion and metastasis.** For example, cleavage of the basement membrane proteins collagen IV and laminin by MMP2 or MMP9 generates novel sites that bind to receptors on tumor cells and stimulate migration.

Answer 211

Locomotion is the final step of invasion, propelling tumor cells through the degraded basement membranes and zones of matrix proteolysis. Migration is a complex, multistep process that involves many families of receptors and signaling proteins that eventually impinge on the actin cytoskeleton. Cells must attach to the matrix at the leading edge, detach from the matrix at the trailing edge, and contract the actin cytoskeleton to ratchet forward. Such movement seems to **be potentiated and directed by tumor cell–derived cytokines,** such as **autocrine motility factors.** In addition, **cleavage products of matrix components** (e.g., collagen, laminin) and some growth factors (e.g., IGFs I and II) have **chemotactic activity for tumor cells.** Furthermore, proteolytic cleavage liberates growth factors bound to matrix molecules. Stromal cells also produce **paracrine effectors of cell motility**, such as **hepatocyte growth factor**–**scatter factor**, which bind to receptors on tumor cells. Concentrations of hepatocyte growth factor–scatter factor are elevated at the advancing edges of the **highly invasive brain tumor glioblastoma multiforme,** supporting their role in motility.

Answer 212

True Stromal cells that interact with tumors include **innate and adaptive immune cells** (discussed later), as well as **fibroblasts**. A variety of studies have demonstrated that tumor-associated fibroblasts exhibit altered expression of genes that encode ECM molecules, proteases, protease inhibitors, and various growth factors. Thus, tumor cells live in a complex and ever-changing milieu composed of ECM, growth factors, fibroblasts, and immune cells, with significant cross-talk among all the components. The most successful tumors may be those that can co-opt and adapt this environment to their own nefarious ends.

Answer 213

first capillary bed available to the tumor.

Answer 214

* • Because the first step in extravasation is adhesion to the endothelium, tumor cells may have adhesion molecules whose ligands are expressed preferentially on the endothelial cells of the target organ. Indeed, it has been shown that the endothelial cells of the vascular beds of various tissues differ in their expression of ligands for adhesion molecules. * • Chemokines have an important role in determining the target tissues for metastasis. For instance, some breast cancer cells express the chemokine receptors CXCR4 and CCR7. [114] The chemokines that bind to these receptors are highly expressed in tissues to which breast cancers commonly metastasize. Blockage of the interaction between CXCR4 and its receptor decreases breast cancer metastasis to lymph nodes and lungs. Some target organs may liberate chemoattractants that recruit tumor cells to the site. Examples include IGFs I and II. * • In some cases, the target tissue may be a nonpermissive environment—unfavorable soil, so to speak, for the growth of tumor seedlings. For example, though well vascularized, skeletal muscles are rarely the site of metastases.

Answer 215

For example, breast cancer metastases to bone are **osteolytic because of** **the activation of osteoclasts** in the **metastatic site**. **Breast cancer cells secrete parathyroid hormone–**r**elated protein (PTHRP),** which **stimulates osteoblasts to make RANK ligand (RANKL).** RANKL then activates osteoclasts, which degrade the bone matrix and release growth factors embedded within it, like IGF and TGF-β. With a better molecular understanding of the mechanisms of metastasis our ability to target them therapeutically will be greatly enhanced

Answer 216

Several competing theories have been proposed to explain how the metastatic phenotype arises. The **clonal evolution model** suggest that, as mutations accumulate in genetically unstable cancer cells and the tumor become heterogeneous ( Fig. 7-38A ), a subset of tumor cell subclones develop the right combination of gene products to complete all the steps involved in metastasis. Thus, metastatic subclones result from clonal evolution, and it is only the rare cell that acquires all the necessary genetic alterations and can complete all the steps. However, recent experiments, in which gene expression profiles of primary tumors and metastatic deposits have been compared, challenge this hypothesis. For example, a subset of breast cancers has a gene expression signature similar to that found in metastases, although no clinical evidence for metastasis is apparent. In these tumors it seems that most if not all cells develop a predilection for metastatic spread during early stages of carcinogenesis. Metastases, according to this view, are not dependent on the stochastic generation of metastatic subclones postulated above. The alternative hypothesis suggested by these data is that metastasis is the result of multiple abnormalities that occur in many, perhaps most, cells of a primary tumor, and perhaps early in the development of the tumor ( Fig. 7-38B and C ). Such abnormalities give most cells within the tumor a general predisposition for metastasis, often called the “metastasis signature.” [116] This signature may involve not only properties intrinsic to the cancer cells but also the characteristics of their microenvironment, such as the components of the stroma, the presence of infiltrating immune cells, and angiogenesis ( Fig. 7-38D ). It should be noted, however, that gene expression analyses like those described above would not detect a small subset of metastatic subclones within a large tumor. Perhaps both mechanisms are operative, with aggressive tumors acquiring a metastases-permissive gene expression pattern early in tumorigenesis that requires some additional random mutations to complete the metastatic phenotype. **A third hypothesis suggests that background genetic variation, and the resulting variation in gene expression**, in the human population contributes to the generation of metastases. In mouse models, cancers induced with the same oncogenic mutations can have very different metastatic outcomes depending on the strain (i.e., background genetics) of the mouse used. Even very strong oncogenes can be significantly affected by background genetics. The fourth hypothesis is a corollary of the tumor stem cell hypothesis, which suggests that if tumors derive from rare tumor stem cells, metastases require the spread of the tumor stem cells themselves

Answer 217

FIGURE 7-38 Mechanisms of metastasis development within a primary tumor. A nonmetastatic primary tumor is shown (light blue) on the left side of all diagrams. Four models are presented: A, Metastasis is caused by rare variant clones that develop in the primary tumor; B, Metastasis is caused by the gene expression pattern of most cells of the primary tumor, referred to as a metastatic signature; C, A combination of A and B, in which metastatic variants appear in a tumor with a metastatic gene signature; D, Metastasis development is greatly influenced by the tumor stroma, which may regulate angiogenesis, local invasiveness, and resistance to immune elimination, allowing cells of the primary tumor, as in C, to become metastatic.

Answer 218

A metastasis suppressor gene is defined as a gene whose loss promotes the development of metastasis **without an effect on the primary tumor.** Accordingly, expression of a metastasis oncogene favors the **development of metastasis without effect upon the primary tumo**r. ``` At least a dozen genes lost in metastatic lesions have been confirmed to function as **“metastasis suppressors”**. [117,] [118] ``` Their molecular functions are varied and not yet completely clear; however, most appear to affect various signaling pathways. Interestingly, recent work has suggested that two miRNAs, mir335 and mir126, suppress the metastasis of breast cancer, while a second set (mir10b) promotes metastasis.

Answer 219

SNAIL and TWIST,