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carcinoma

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Breast carcinoma

• The gene pik3CA, which codes for Phosphati-dylinositol 3-Kinase P110a, is amplified in a portion of cervical and ovarian tumors.

• The copy number of the urokinase plasminogen activator (upa) gene is often increased in hormone refractory prostate cancer.

• Amplification of the chromosome locus 2p21 occurs in about 30% of thyroid neoplasms. This causes a rearrangement and amplification of the pkce gene and results in the overexpression of a chimeric truncated pkce mRNA, coding for the NH2-terminal amino acids 1-116 of the enzyme form, fused to an unrelated sequence. Cells expressing the truncated PKCe are resistant to apoptosis. This is associated with higher BCL-2 levels, a marked impairment in P53 stabilization, and dampened expression of BAX [Knauf et al. 1999].

• In esophageal squamous carcinoma, amplification of 11q21-q23 frequently occurs. This leads to overexpression of the antiapoptotic gene ciap1 [Imoto etal. 2001].

The deletion of one allele of a tumor suppressor gene (loss of heterozygosity) [Cavenee et al. 1983] leads to symptoms if the remaining allele is mutated. Losses of heterozygosity, i.e., losses of a maternal or paternal allele in a tumor, are widespread and are often accompanied by a gain of the opposite allele. On average, cancers of the colon, breast, pancreas, or prostate may loose 25% of their alleles. It is not unusual for a tumor to have lost over half of its alle-les. Haplo-insufficiency (allelic insufficiency) describes a phenotype resulting from the loss of one functional allele of a given gene in diploid cells.

• Deregulated signaling through Phosphatidylinositol 3-Kinase is common in glioblastoma and in advanced prostate carcinoma. This may be due to loss of heterozygosity at 10q23. It causes the loss of pten, which is highly correlated with activation of PKB (AKT). The elevated PKB activity leads to phosphorylation of mTOR and of the Forkhead transcription factors (FOXO1, FOXO3a, and FOXO4). The loss of one copy of pten is also sufficient to promote tumor progression in germ cell, gonadostromal, thyroid, and colon tumors.

• The WNT signaling pathway is often up-regulated in epidermal cancers. The gene for the APC homolog apc-2 (apcl) is located on chromosome 19p13.3, a region that is commonly lost in ovarian cancer. High frequency apc-2 allelic imbalance in ovarian cancers implies that APC-2 may act as a tumor suppressor in this type of malignancy.

• A 600 kb region on chromosome 8p22-p21.3 is commonly deleted in hepatocellular carcinoma, colorectal carcinoma, and non-small cell lung carcinoma. This reflects a loss of prlts (PDGFR-p-like tumor suppressor) [Fujiwara et al. 1995].

• The endonuclease MUS81 (EME1, MMS4) {11q13} plays a role in processing stalled DNA reduplication forks. Monoallelic loss of the gene encoding MUS81 increases the susceptibility to chromosomal damage and constitutes a profound predisposition to lym-phomata [McPherson et al. 2004].

• The transcriptional activator DMP1 regulates the expression of p14ARF. Its down-regulation through haplo-insufficiency increases the susceptibility to lymphoma [Inoue et al. 2001].

• cdkn1B, the gene encoding P27KIP1, is haplo-insufficient for tumor suppression [Fero et al. 1998] in pituitary adenomata and various epithelial cancers.

3.4.2 Deletion of functional domains

The most common mode of activation of receptor tyrosine kinase proto-oncogene products is the deletion of the NH2-terminal ligand-binding domain, which forms a constitutively active kinase (erbB, erbB2, ros, met, ret, trk). The deletion of sequences at the extreme COOH-terminus may also activate kinase activity (erbB, fms, kit, ret).

The activity of the SRC family nonreceptor tyrosine kinases (SRC, YES, FGR, LCK, FYN, LYN, HCK) is negatively regulated by tyrosine phsophory-lation in the COOH-terminal portion of the molecule, the deletion of which can be a transforming event. Likewise, deletion or mutation of the NH2-terminal SH3 region may activate transforming potential. Deletion of the SH3 region of ABL is sufficient to confer the potential for transformation [van der Plas et al. 1991]. Deletion of the NH2-terminal regulatory domain in the serine/threonine kinase RAF results in constitutively up-regulated activity in the COOH-terminal catalytic domain [Cooper 1999].

Deletions may occur outside the coding regions of genes, including introns or promoters. While these do not affect the structure of the encoded proteins, they may alter gene expression or RNA stability.

• Homozygous deletion may occur in the nf1 gene in neurofibrosarcoma, pheochromocytoma, and astrocytoma with the consequence of dysregulated P21RAS and unrestricted cell cycle progression.

• Deletion of the NH2-terminal ligand-binding domain of KIT occurs in gastrointestinal stromal tumors and in mast cell tumors.

• Ligand-independent activation may occur if MET is truncated. This results in its cytoplasmic, rather than transmembrane localization. The subcellular localization of MET contributes to determining its carcinogenic potential. Cytoplasmic MET may be tumorigenic in the breast.

• Several cd95 gene mutations occur in myeloma and T-cell leukemia. They include deletions that lead to truncated forms of the death receptor. These mutated forms of CD95 might interfere in a dominant negative way with apoptosis induction by wild-type CD95.

• A truncating mutation in SRC at codon 531 arises in about 12% of cases of advanced colon cancer [Irby etal. 1999].

• Genetic mutations leading to a truncated or unstable PTC protein are associated with familial or sporadic basal cell carcinoma.

• In familial and sporadic meningiomata, a deletion in the fifth intron of the sis gene may occur. The intact sis gene has an Alu sequence in this region, which includes two perfect 130 nucleotide-repeated sequences, separated by five base pairs. The deleted allele in a fraction of meningioma cases misses one copy of the 130 base pair repeats and the intervening five base pairs [Bolger et al. 1985; Smidt etal. 1990].

3.4.3 Point mutations

Spontaneous point mutations may lead to the continuous activation of proto-oncogene products or inactivation of tumor supressor gene products [Cooper 1999]. Point mutations in proto-oncogenic receptor tyrosine kinase genes may lead to confor-mational changes in the encoded protein that mimic an activated state, without deletion or loss of ligand binding (fms, erbB2). The constitutive activation leads to ligand-independent signal transduction and results in excessive cell cycle progression.

Mutations of the p53 gene occur in tumors of the brain, breast, lung, colon, and mesenchyme [Nigro 1989]. Tumor-associated mutations in p53 are predominantly point mutations that result in single amino acid substitutions (Figure 3.4.3.A). This is distinct from many other tumor suppressor genes, in which large deletions or frameshift mutations tend to result in the complete loss of protein expression. Several types of mutant p53 exist, according to the sites of mutation and the resulting phenotypes [Michalovitz et al. 1991]. Certain p53 codons, encoding the residues 175, 245, 248, 249, 273, and 282, have a dysproportionately high mutation frequency. Point mutations in p53 frequently occur in the DNA-binding region. More than 95% of alterations in the p53 gene are point mutations that produce a mutant protein, which has lost its transactivational activity,

- Null mutations with totally inactive P53 that do not directly intervene in transformation.

- Dominant negative mutations with a totally inactive P53 that is still able to interfere with wild-type P53 expressed from the wild-type allele; many tumor cells retain the ability to express mutant P53 proteins that may be more stable than wild-type P53 and may act as dominant negative inhibitors.

- Dominant positive mutations, where the normal function of P53 is altered, but in this case the mutant P53 acquires an oncogenic activity that is directly involved in transformation. In paticular, some P53 mutants exhibit a trans-dominant phe-notype and are able to associate with wild-type P53, expressed by the remaining wild-type allele, to induce the formation of an inactive het-erooligomer [Halevy et al. 1990; Milner and Medcalf 1991].

Figure 3.4.3.A. P53 mutations in cancer. P53 is a 393 residue protein that contains a NH2-terminal transactivation domain, a proline-rich SH3 ligand, a core DNA-binding domain (also a noncanonical SH3 ligand), a tetramerization domain and a COOH-terminal regulatory domain. Five boxes displaying the regions of greatest sequence conservation are shown, four map to the core domain. A histogram of P53 missense mutations shows that 95% of mutations occur in the core domain, six labeled residues are hot spots for mutation. [Reproduced from Bullock and Fersht 2001. With permission from Macmillan.]

Figure 3.4.3.A. P53 mutations in cancer. P53 is a 393 residue protein that contains a NH2-terminal transactivation domain, a proline-rich SH3 ligand, a core DNA-binding domain (also a noncanonical SH3 ligand), a tetramerization domain and a COOH-terminal regulatory domain. Five boxes displaying the regions of greatest sequence conservation are shown, four map to the core domain. A histogram of P53 missense mutations shows that 95% of mutations occur in the core domain, six labeled residues are hot spots for mutation. [Reproduced from Bullock and Fersht 2001. With permission from Macmillan.]

In loss of heterozygosity, one allele is deleted, which typically leads to symptoms if the affected gene is a tumor suppressor genes and the remaining allele is mutated. Point mutations of p53 in cancer are frequently accompanied by heterozygous loss of the short arm of chromosome 17. The majority of colorectal carcinomata have mutations in p53. Frequently, mutations in one allele of p53 are associated with deletions of chromosome 17p [Baker et al. 1989].

• Constitutively activated versions of the CSF-1 Receptor tyrosine kinase c-FMS contain single point mutations (codons S301L and F969Y in 10-20% of AML or myelodysplastic syndrome). Like the v-FMS oncogene product, receptors bearing the activating mutations retain high affinity binding sites for CSF-1, but are retarded in their transport to the cell surface and are phosphorylated on tyrosine in the absence of ligand [Roussel et al. 1988]. Furthermore, transforming point mutations of FMS are often associated with reduced receptor degradation [Morley et al. 1999]. c-CBL binds to a phosphotyro-sine at the COOH-terminal end of c-FMS. This leads to ubiquitination of FMS [Mancini et al. 2002].

• Constitutively activated versions of a normal receptor tyrosine kinase, the c-KIT Receptor, are generated by single point mutations. Gain-of-function mutations in c-kit, leading to constitutive activation of the KIT Receptor, specifically the D814Y mutation in the cytoplasmic kinase domain, are associated with myeloproliferative disorders (mastocytomata)

[Piao and Bernstein 1996]. A point mutation affecting the catalytic region in the kinase domain of the KIT protein leads to spontaneous transformation of melanocytes [Larue et al. 1992].

• In neuroblastomata, the erbB2 (her-2/neu) onco-gene may have a point mutation resulting in the alteration of a single amino acid in the transmembrane region of the receptor. This causes its constitutive activation. The erbB2 gene is also activated by a point mutation encoding V664E, which may result in cell transformation in breast cancer, bladder cancer, colon cancer, lung cancer, and gastric cancer. Point mutations in the transmembrane domain of ERBB2 enhance its transforming properties. They may have a stabilizing effect on the conformation, which results in receptor dimerization and activation.

• A point mutation S267P, in the extracellular third Immunoglobulin-like domain, and a splice site mutation 940-2A^G of the fgf receptor-2 gene occur in gastric carcinoma.

• Point mutations in met occur in hereditary and sporadic papillary renal carcinomata [Schmidt et al. 1997], hepatocellular and gastric carcinomata [Park et al. 1999; Lee et al. 2000], and head and neck squamous carcinomata [Di Renzo et al. 2000]. Point mutations in the kinase domain convert MET to an oncogenic receptor. Such mutants are catalytically highly active, which correlates with more efficient MET autophosphorylation and phosphorylation of substrates. The constitutive binding of c-SRC to the cytoplasmic domain of the MET M1268T mutant in renal papillary carcinomata, elevates c-SRC phospho-rylation and activity. MET M1268T also phosphory-lates substrates of the cytosolic kinase c-ABL, whereas wild-type MET does not. The expression of MET M1268T induces P-Catenin tyrosine phospho-rylation and accumulation, induces constitutive activation of the transcription factor TCF, which acts in concert with P-Catenin in the nucleus to increase the expression of the target genes myc and cyclin D1.

• Germline activation of the ret gene, attributable to specific point mutations, causes medullary thyroid carcinoma, a neoplastic transformation of the Calcitonin secreting thyroidal C-cells. Mutations of ret may also cause multiple endocrine neoplasia type 2 (C634R in MEN2A, M918T in MEN2B).

• Consecutive to missense mutations, the Androgen Receptor may loose its ligand specificity and promiscuously respond to a range of steroid hormones and pseudo-androgens. Receptors with the T877A mutation are stimulated by the antagonist flutamide. The double mutant T877A, L701H, called ARCCR (cortisol and cortisone responsive), has increased affinity for glucocorticosteroids. The H874Y mutation influences the binding of coactivator proteins by affecting the conformation of helix 12.

• Missense mutations of the Estrogen Receptor that substitute tyrosine 537 in the ligand-binding domain for asparagine occur in metastatic breast cancer [Zhang et al. 1997]. The lysines 302 and 303 are principal substrates for acetylation by CBP. The missense mutation L303R occurs frequently in premalignant lesions of the breast.

• Point mutations leading to ras oncogene activation are frequently induced by chemical carcinogens. Point mutations that convey transforming potential to RAS occur in the guanine nucleotide-binding pocket and either decrease the GTPase activity or increase the exchange rate of bound GDP for free GTP.

• ARMET (Arginine-Rich Mutated in Early Stage Tumors, Arginine-Rich Protein, ARP) [Shridhar et al. 1996] is a 234 amino acid arginine-rich protein. The gene is located in 3p21.1 and spans about 600 kb. The multiple arginine-encoding area of the gene is subject to a high frequency of genetic variation. At the cytogenetic level, the region containing armet is frequently deleted in a variety of solid tumors, although not in pancreatic cancer. A specific mutation of armet, changing codon 50 from ATG to AGG (M50R) or deletion of codon 50 of the armet gene occurs in various tumor types [Shridhar et al. 1997a], including renal carcinomata, as do mutations involving codon 50 in pancreatic cancers [Shridhar et al. 1997b]. Either of the changes abolishes a methionine residue and gives rise to an uninterrupted string of AGG trinucleotides, encoding arginines in its predicted protein product. Four other nucleotide substitutions in codon 50 that replace methionine with four different amino acids, other than arginine, may occur, suggesting that loss of this methionine residue is critical to a carcinogenic role of ARMET. Furthermore, a mutation AGG to AAG (arginine to lysine) in the adjacent codon 51 can occur in tumors, reflecting the importance of this region to transformation. Only a single copy of the armet gene is mutated in the cancer cells, indicating its possible causal role as an oncogene.

• A single base change in the tumor suppressor gene apc, due to a polymorphism, replaces a thymine with an adenine, generating a stretch of eight adenines. Such sequences are often misread by polymerases, causing consecutive somatic frameshift mutations, which then pose a predisposition for malignant transformation to colorectal cancer [Laken et al. 1997].

• Li-Fraumeni syndrome may be caused by a R145W missense mutation in CHK2 that destabilizes the encoded protein. The half-life of the mutated CHK2 is reduced due to degradation in the protea-some pathway. [Lee et al. 2001]. In the absence of CHK2, the S phase checkpoint is impaired and cell cycle progression is facilitated.

• Inactivating germline point mutations in cdkn2A, encoding the tumor suppressors P16INK4a and P14ARF, predispose to the familial atypical multiple mole melanoma (FAMMM) syndrome, but also occur in rare families with clustering incidence of cutaneous malignant melanoma (CMM). They include M53V, M53I, G101W, G122V, and V126D. The M53V mutation occurs in exon 2, where p16 and the alternative reading frame for p14ARF both share transcript sequences, and is coupled to a D67G alteration in P14ARF. In contrast, the M53I mutation is coupled to a distinct D68H alteration in p14ARF [Yang et al. 2004]. The G122V variant retains some capacity to interact with CDKs, yet it is significantly impaired in its ability to cause G1 cell cycle arrest. In hereditary melanoma, a G^T transversion mutation in the last nucleotide of exon 2 affects the aspartate residue at position 153 of P16INK4a. This mutation, D153spl(c.457G > T), and a related mutation at the next nucleotide, IVS2 + 1G > T, result in identical aberrant splicing. The alternate splice products for p16INK4a and p14ARF include a 74 bp deletion in exon 2, revealing a cryptic splice site, and completely skip exon 2 [Rutter et al. 2003].

• A high incidence of cd95 mutations exists in bladder cancer. In codon 251, the mutation G993A is a hot spot in this malignancy [Lee et al. 1999b]. Several cd95 gene mutations occur in myeloma and T-cell leukemia [Cascino et al. 1996]. They include point mutations in the cytoplasmic death domain of CD95. These mutated forms of CD95 may interfere in a dominant negative way with apoptosis induction by CD95.

• Missense mutations in bak may occur in gastric and colorectal cancers. They arise only in advanced stages of the disease [Kondo et al. 2000].

3.4.4 Frameshifts

Frameshift mutations are genetic alterations that insert into a DNA sequence or delete from it a number of nucleotides that is not evenly divisible by 3. Due to the triplet nature of gene expression by codons, the insertion or deletion can disrupt the grouping of the codons, resulting in a completely different translation from the original.

Frameshift mutations occur at simple repeat sequences in tumors of the microsatellite mutator phenotype. Microsatellites are short repetitive sequences, which are often copied incorrectly by DNA polymerases because the template and daughter strands in these regions are particularly prone to misalignment. These replication-dependent events create loops of extrahelical bases, which would produce frameshift mutations unless reversed by mismatch repair. Germline mutations in mismatch repair genes are associated with hereditary nonpolyposis colon cancer. Microsatellite instability and the associated frameshift mutations in genes also arise in sporadic colon, gastric, endometrial, and ovarian tumors.

• A large portion of lobular breast carcinomata and gastric carcinomata contain E-cadherin frameshift mutations. This leaves the E-Cadherin protein truncated and unable to mediate adhesion. The truncation mutants may exert a dominant negative effect on other Cadherins.

• In colorectal tumors with microsatellite instability approximately 40% exhibit one base pair deletion, resulting a frameshift mutation in a tract of nine adenosines within the coding region of the tcf-4 gene, a crucial member of the WNT^APC pathway [Duval et al. 1999].

• Genes containing repetitive sequences within their coding regions can be targets for microsatellite insta bility tumorigenesis. Frameshift mutations occur in bax in about 50% of colon carcinomata with microsatellite instability. They typically affect a tract of eight deoxyguanosines, spanning the codons 38-41 in the third coding exon [Rampino et al. 1997].

• Frameshift mutations of bax that lead to a loss of its expression are common. Tumor cells with these frameshift mutations are more resistant to apopto-sis. Reduced BAX expression may be associated with shorter survival in breast adenocarcinoma [Krajewski et al. 1995].

• prlts (PDGFR-p-like tumor suppressor) is a tumor suppressor gene located on chromosome 8p22-p21.3. Frameshift mutations of prlts arise in hepa-tocellular carcinoma and colorectal cancer [Fujiwara et al. 1995].

• A frameshift mutation in tgf-fi receptor 1 frequently occurs in ovarian cancer. Frameshift mutations also arise in type II TGF-ft receptor in colorectal cancer. They affect a tract of ten adenosines in the coding sequence. A high rate of mutations occurs in tumors at Dukes B stage, showing a great extent of vascular invasion. The frameshift mutations in the TGF-P Receptors inactivate their growth controlling functions.

3.4.5 Chromosome translocations

Translocations involve the exchange of material between chromosomes. Such structural rearrangements at the molecular level can juxtapose segments of DNA that are not normally adjacent to one another. Frequently, these juxtapositions are very precise, with the exchange point in one or both participating chromosomes being positioned within a few base pairs. Balanced translocations generate derivative chromosomes with no apparent loss or gain of sequences from either chromosome. Unbalanced translocations are associated with the loss of sequences from the involved chromosomes. Nonreciprocal translocations are transpositions of two segments between nonho-mologous chromosomes with loss or gain of genetic material as the result. Inversions involve only one chromosome, in which two breaks occur and the intervening segment is rejoined in an inverted manner. There is no loss or gain of chromosomal material. An inversion is paracentric if the inverted segment is on the long or short arm of the chromosome and does not include the centromere. The inversion is pericen-tric if breaks occur on both, the short arm and the long arm, and the inverted segment contains the centromere (Table 3.4.5.A).

Table 3.4.5.A. Chromosome translocations in hematologic malignancies. Chromosome translocations are a common cause for the transformation of blood cells or their precursors. Typically, they either form chimeric proteins or place proto-oncogenes under the control of highly active, lineage specific promoters. ALCL = anaplastic large cell lymphoma, AML = acute myeloid leukemia, ABL = acute basophilic leukemia (a rare type of acute myeloid leukemia), ANLL = acute nonlymphocytic leukemia, ALL = acute lymphocytic leukemia, CLL = chronic lymphocytic leukemia, CML = chronic myeloid leukemia, MALT = mucosa-associated lymphoid tissue

Oncogene

Fusion partner

Consequence

Malignancy c-myc {8q24} c-myc {8q24} c-myc {8q24} pax5 {9p13} bcl-l {1q13} bcl-2 {l8q21} bcl-lO {1p22} bcl-ó {3q27}

bcl-ó {3q27} bcl-ó {3q27} bcl-ó {3q27} bcl-ó {3q27} bcl-ó {3q27} bcl-lla {2q13-15} abl {9q34}

igk {2p12} igl {22q11} igh {14q32} igh {14q32} igh {l4q32} igh {l4q32} igh {l4q32} igh {l4q32}

igk {2p12} igl {22q11} rhoH {4p13} obfl {11} lcpl {13q14} igh {14q32} bcr {22q11}

chic2 {4q11—12} c-myc {8q24} tcll {14q32.1} mtcpl {Xq28} lmol (rhoml, ttgl) {11p13} lmo2 (rhom2, ttg2) {11p13} hoxll {10q24} lyll, hoxll, tall, tal2, or lmo2 tcll {14q32.1} tall {1p32} tall {1p32} e2A (ig enhancer binding factor) {9p13} e2A {9p13}

jak2 {9p24} abl {9q24.1} pdgfr {5q31-q32} pax5 {9p13} rara {17q11.2-12} rara {l7q11.2-12} rara {l7q11.2-12} rara {l7q11.2-12} rara {l7q11.2-12}

etvó {12p13} tcralS {14q11} tcralS {l4q11} tcralS {l4q11} tcralS {l4q11}

tcralS {14q11}

tcralS {14q11} tcralS or tcrß

tcrß {7q25} tcrß {7q25} tcta {3p21} prl (homeobox gene) {1q23} pbxl {1q23}

hlf {17q22} il-2 {4q26-q27} tel (etvó, ETS family transcription factor) {12p13} tel {12p13} tel {l2p13} tel {l2p13} tel {l2p13} pml {15} Plzf {11} npm {5} numa {11q13} stat5B {17q11.2}

Fusion protein Fusion protein Fusion protein

Promoter exchange Excessive bcl-6 expression

Promoter exchange

"Philadelphia chromosome translocation" Oncogenic Tyrosine Kinase Fusion protein containing ABL-2 Kinase plus ETV6 HLH Oligomerization Domain

Constitutive PKB activation

Constitutive PKB activation Fusion Protein

Oncogenic transcription, specific binding to ig enhancer Oncogenic transcription

Oncogenic Tyrosine Kinase Oncogenic Tyrosine Kinase Oncogenic Tyrosine Kinase

Transcriptional repressor Transcriptional repressor

Burkitt lymphoma

Burkitt lymphoma

Burkitt lymphoma

B-cell non-Hodgkin lymphoma

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