Molecular alterations in sporadic breast cancer

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1 Critical Reviews in Oncology/Hematology 44 (2002) Molecular alterations in sporadic breast cancer F. Lerebours *, R. Lidereau E0017 INSERM/Oncogénétique, Centre René Huguenin, 35 rue Dailly, F St-Cloud, France Accepted 14 August 2001 Contents 1. Tumorigenesis: a multi-stage process Alterations observed in breast cancer DNA content and cell cycle Genomic regions most frequently altered in breast cancer DNA Gain of genetic material Loss of genetic material Microsatellite locus instability Chromosomal instability; chromosome segregation anomalies during mitosis Patterns of genes expression Genes involved in breast tumorigenesis Amplification and overexpression of oncogenes in breast cancer Inactivated genes, tumor suppressor genes Inactivation mechanisms Main tumor suppressor genes implicated in breast cancers Other genes Function of implied genes Transduction pathways Cell cycle Apoptosis, senescence Repair Tumor dissemination Is there a sequence of the genetic events? Applications for diagnosis, prognosis and treatments Hereditary breast cancer Prognostic markers Predictive markers Targets for therapy Reviewers Acknowledgements References Biography * Corresponding author. Tel.: ; fax: address: f.lerebours@stcloud-huguenin.org (F. Lerebours) /01/$ - see front matter 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S (01)

2 122 F. Lerebours, R. Lidereau / Critical Reiews in Oncology/Hematology 44 (2002) Abstract Breast cancer is a genetic disease. Like other human cancers, it is thought to occur as the result of progressive accumulation of genetic aberrations. These aberrations result in a deviation of the gene expression profiles from that of the normal progenitor cell. In up to 99% of cases, breast cancer is due to solely somatic genetic aberrations without germ-line ones. Considerable progress have already been made in understanding the genetic mechanisms underlying the development and progression of breast cancer. Several extensively studied genes are now well known to be involved. Unfortunately, our ability to make clinically useful interventions on the basis of these data is limited. Because of the involvement of multiple genes and complex pathways in a single cancer cell, the molecular dysfunctioning underlying breast cancer remains to be completely clarified. In a next future, studying the global gene expression of different types of tumors will allow the development of expression profiles unique for a breast cancer, its stage and prognostic category, leading to diagnostic assays and the identification of new therapeutic targets Elsevier Science Ireland Ltd. All rights reserved. Keywords: Breast cancer; Genetic alterations; Oncogene; Tumor suppressor gene 1. Tumorigenesis: a multi-stage process The relation between the occurrence of a cancer and the existence of genetic alterations is now well established. In breast cancer as in the majority of cancers, these alterations are mostly somatic. In 4 10% of breast cancers, a germ-line mutation will predispose to breast cancer. This mutation seems to help the occurrence of specific somatic genetic alterations, which will lead to the cancer phenotype. The genetic alterations observed in the course of the malignant transformation are varied: chromosomal deletions, translocations, amplifications and rearrangements, duplications or whole chromosome losses (aneuploidy), and point mutations. Some of these alterations lead to the modification of the expression and/or the structure of the product of the gene implied by these alterations. It will result in: activating genes in a dominant way: the alteration of only one of the two copies of the gene is required and these genes have been called oncogenes. inactivating genes whose loss of function participates to tumorigenesis; these genes have been called tumor suppressor genes. activating or inactivating genes whose products interfere with the genome stability: genes regulating the processes of mitosis and DNA repair enzymes. The alteration of these genes will help the occurrence of alterations of the more directly implicated genes (i.e. oncogenes and tumor suppressor genes). finally, the above mentioned gene expression modifications can result, not from the direct alteration of the gene itself, but from alteration of genes acting on the modeling of the chromatin or on the DNA methylation status. Such mechanisms are called epigenetic, because unlike mutations, the modifications may be reversible. The activation or inhibition of only one of these genes is not sufficient to convert the normal phenotype of a cell into a tumor one: multiple independent mutational events are required. The accumulation of these genetic alterations, which interact and cooperate between themselves, is necessary to lead to the tumor phenotype. During this multi-stage process, each new genetic alteration acquired by the cell will give it new properties, favoring the selection of this clone and thus converging to the tumoral phenotype. One to 5% of the approximately human genes has an altered expression in the tumor cells of a single cancer. The altered genes are generally not specific to a histological type of cancer. The understanding of this complex phenomenon which is carcinogenesis requires the identification of these numerous genetic alterations or mutations, of which all or part of it is responsible for the initiation and the tumor progression. In due time, the identification of the responsible genetic alterations will have repercussions on diagnosis, prognosis and therapeutic. 2. Alterations observed in breast cancer Somatic genetic alterations have been studied in multiple ways: (1) flow cytometry measures quantitative DNA anomalies; (2) cytogenetics anomalies have been first reported by karyotype analysis; (3) molecular cytogenetic like CGH, FISH and more recently CGH arrays, has refined the analysis of cytogenetic anomalies; (4) molecular biology techniques detect mutations and expression modifications of genes. To this date, few genes have been found mutated in breast cancer. However, two types of recurrent molecular anomalies suggest either the mutation of non-identified genes, or the mutation of known genes, but whose mutation has not been detected by means of the employed techniques. Recurrent LOH or amplifications. Increased or decreased gene expression at the RNA or protein level DNA content and cell cycle Approximately 70% of breast cancers are aneuploid, with a DNA index (ratio between the DNA content of

3 F. Lerebours, R. Lidereau / Critical Reiews in Oncology/Hematology 44 (2002) a tumor cell in G0/G1 and a normal cell in G0/G1) ranging between 1.2 and 1.9 [1]. The percentage of highly proliferative breast cancers as demonstrated by a high tumor cell S-phase fraction, varies from one study to another. These variations are partly due to the chosen threshold value, which ranges from 2.6 to 4% for diploid tumors, and from 7.6 to 17.3% for aneuploid tumors [2]. A high rate of tumor proliferation has often been correlated to a higher histological grade and aneuploidy Genomic regions most frequently altered in breast cancer DNA DNA amplification suggests the existence within the amplified region of dominantly acting genes. Conversely, recurrent DNA losses may point to tumor suppressor genes. As well as gene alterations, the implied chromosomal regions are often common to other histologic types of cancer [3,4]. Two methods have been mostly used to emphasize these anomalies: Allelotyping: the comparison of allelic status at chosen loci in tumor and normal DNA by the use of PCR with highly polymorphic markers has been named allelotyping. The histogram on Fig. 1 gives the average of LOH frequencies per chromosomal arm reported in breast cancer allelotypes. CGH: CGH involves in situ hybridization of normal and tumor DNA to normal metaphase chromosomes. Each DNA is labeled by a different modified nucleotide and revealed by specific antibodies labeled with two different fluorochromes. Actually CGH is frequently performed using directly fluorochrome-conjugated DNAs. Increases or decreases in the fluorescence ratio along the chromosomes indicate regions of losses and gains in the tumor DNA. The histogram of Fig. 2 gives the average of amplifications and deletions in breast cancer CGH studies. CGH-array is a more recent technique, with promising results [5]. This technique allows refining the regions that are lost or amplified in the tumor DNA Gain of genetic material Amplifications can involve short chromosomal regions or entire chromosome arms. These different kinds of amplifications result from distinct genetic mechanisms. The gain of entire chromosome arms may be due to mitotic non-disjunctions, whereas gene amplification could reflect the selective pressure resulting from the dominant action of some genes. Whole chromosome gains usually lead to low rates of amplification (2 5 times) but for hundreds of genes, against times for gene amplifications. In that case, amplification is generally associated to an overexpression of the gene(s) product of the amplified region. Gain of entire chromosome arms Cytogenetic analysis and CGH have shown frequent gain of 1q and 8q [4,6 8]. Amplified regions Besides the 1q (in particular in 1q21, 1q32, 1q41) and the 8q (in 8q24) amplified in approximately 40% of cases, the most frequently amplified regions in breast cancer are the 11q13 band, the 16p11, 17q11.2, 17q24 and 20q13 regions, in over 15 20% of breast cancers [7 17]. Fig. 2 shows the mean amplification frequency for each chromosomal arm reported in CGH studies. Very few known genes are clearly implicated in these amplifications, and thus numerous genes are still to be identified (Table 1). Fig. 1. Percentage of LOH per chromosomal arm in breast cancer.

4 124 F. Lerebours, R. Lidereau / Critical Reiews in Oncology/Hematology 44 (2002) Fig. 2. CGH studies: amplifications and deletions in breast tumors Loss of genetic material Chromosomal deletions are very frequent events observed in breast cancer cells. Their recurrence from one tumor to another at the same loci suggests the existence at these loci of tumor suppressor gene(s). Indeed the loss of function of TSGs generally requires an inactivation of each parental copy of the gene, most often by mutation of one allele and LOH of the wild type one [18]. The histograms in Figs. 1 and 2 show the mean frequency of deletions observed respectively in allelotyping and CGH studies. Despite several discrepancies in the frequencies depending on the approach, these histograms point to the most frequently lost chromosomal arms: 1p, 3p, 6q, 8p, 9p, 11p, 13q, 16q, 17p, observed in more than 20 25% of breast cancers [19,20]. Deletions at 1q, 7q, 11p, 17q, 18q and 22q have been reported in 15 20% of breast cancers by LOH studies, and less frequently by CGH. The discrepancies between allelotypes and CGH have several possible reasons: (1) The detection of small size deletions (i.e. inferior to 10 Mb) may escape to CGH. (2) Allelic imbalances observed in LOH studies may delineate either an allele gain or a deletion of the other allele. CGH enables to distinguish these two phenomenon s. (3) In LOH studies, focusing on chromosomal regions frequently deleted in other cancers or containing known TSGs has created a bias. The systematic study of the whole genome is necessary to compare allelotyping and CGH results. Table 1 lists the genomic regions recurrently lost in breast cancers and the candidate genes implicated in those regions Microsatellite locus instability The mutation of DNA mismatch repair genes (MLH1, MSH2 PMS1, PMS2, MSH6, and MSH3) has for consequence an MSI (microsatellite instability) tumor phenotype (also called RER for Replication Error) characterized by a genetic instability at microsatellite loci. This phenotype is found in tumors which patients belong to HNPCC families. Moreover, 15 20% of sporadic colorectal cancers are of MSI phenotype [21]. This phenotype is not accompanied by chromosomal instability (i.e. LOH, amplifications, rearrangements) nor aneuploidy [22]. Besides rare breast cancers occurring in HNPCC families, genomic instability pathways of carcinogenesis characterized by MMR defects and MSI, does not appear to play a role in the genesis of breast cancer [23] Chromosomal instability; chromosome segregation anomalies during mitosis Chromosomal instability and aneuploidy are frequently observed in breast cancers. This phenotype has been correlated in some cases to the loss of control of the mitotic process and to an abnormal segregation of replicated chromosomes [24]. The genes responsible for these controls were first identified in yeast, and are being identified in human (BUB genes). It is likely that the alteration of these genes plays a major role in breast carcinogenesis [25]. Several other genes are involved in the genomic stability: TP53 also controls the chromosomes segregation during mitosis [26]. AT, BRCA1 and BRCA2, and telomerase gene, have also more or less a direct role in genomic stability Patterns of genes expression Several techniques have been developed to analyze known cancer genes expression in tumor DNA. Among these, immunohistochemistry has been widely used. Two complementary methods, cdna microarrays and real-time quantitative PCR will help to analyze the expression level of thousands of genes in a

5 F. Lerebours, R. Lidereau / Critical Reiews in Oncology/Hematology 44 (2002) reliable and reproducible way. In Table 2 are figured the main genes over- or underexpressed in breast cancer in relation with the degree of expression of nearby normal cells. This suggests their role in breast carcinogenesis. The demonstration of mutations of these candidate genes would assert this role. It is noted that some of these genes are localized in amplified or deleted regions reported in Table 1. There exists also gene overexpressions without amplification of their locus, and expression decrease without deletion of the genes locus. The list Table 2 is non-exhaustive, as it is estimated at 1000 the number of genes whose expression is abnormal in breast cancer. 3. Genes involved in breast tumorigenesis 3.1. Amplification and oerexpression of oncogenes in breast cancer ERBB2, HER2/neu: this gene encodes a transmembrane receptor with tyrosine-kinase activity of the ERBB family: ERBB1 (EGFR, 7p13), ERBB2 (17q11.2), ERBB3 (12q13) and ERBB4 (2q33). The transforming potency of these genes is due to amplification and/or overexpression of their products. All are over-expressed in a variable percentage of Table 1 Amplified or deleted chromosomal regions in at least 20% breast cancers, and candidate genes in these regions. Chromosome % Alteration Regions References Candidate genes References 1p 29 Deletion 1p36 [27 31] p73, MPB1 [78 80] 1p31 p35 MDGI [81] 1p13 p22 hrad54 [82,83] 1q 50 Amplification 1q41 44 [32] 1q21 [7,9] MUC1 [84] 20 Deletion 1q21 q25 [20,28,30] 3p 30 Deletion 3p24 p26 [33 36] VHL [85] 3p21 WNT5A [86] 3p13 p14 FHIT [87,88] 6q 30 Deletion 6q13 q21 6q21 q23.3 [19,37 41] 6q25 27 ESR, IGF2R, ZAC [41,89 92] 7q 20 Deletion 7q31 [42,43] MET [93] 8p 40 Deletion 8p22 p23 [20,44 46] FEZ [94] 8p21 NKX3.1 [95] 8q 40 Amplification 8q24 [8,10,47] MYC [96] 9p 25 Deletion 9p21 p22 [48 50] p16/ink4a, p19arf [97] 9p23 p24 [51] 11p 25 Deletion 11p15 [52 54] WT2, NAP2, KIP2 [98] 11q Deletion 11q22 q23 [52,55 57] ATM [99,100] 30 11q24 q25 [58] 20 Amplification 11q13 [14,59,60] CCND1, EMS1 [101,102] 13q 28 Deletion 13q14.1 [61,62] RB1 [103] 13q12 q13 [63] BRCA2 [104] 16p 20 Amplification 16p11 [8,16] 16q 50 Deletion 16q22.1 [64,68] CDH1 [ ] 16q21 q23.3 BCAR1 [108] 16q24.3 CDH13 [109] 17p 49 Deletion 17p13.3 [69,70] HIC1 [110] 17p13.1 [71,72] TP53 [111,112] 17q 32 Deletion 17q12 q21 [72,73] BRCA1, NME1 [ ] 17q22 q24 17q25 qter 17q11 q12 23 Amplification 17q23 24 [8,13] ERBB2, TBX2 [ ] 18q 23 Deletion 18q21.3 q22 [74,75] DCC, maspin [122,123] 20q 20 Amplification 20q13 [8,11,15] AIB1, STK15, ZNF217 [ ] 22q 21 Deletion 22q13 [76,77]

6 126 F. Lerebours, R. Lidereau / Critical Reiews in Oncology/Hematology 44 (2002) Table 2 Main genes of abnormal expression in breast cancer Genes Loci Modification % References Transduction pathways EGFR 7p13 Overexpression [127,128] ERBB2 17q21 Overexpression [119,129] ESR 6q25 27 Underexpression nr [130,131] FGFR1 8p11.2 Overexpression 20 [132] FGFR4 5q35 Overexpression 30 IGF1R 15q25 26 Overexpression 30 [133,134] Cell cycle ATM 11q22 23 Underexpression nr [99,100] TP53 17p13.1 Overexpression a [112,135] RB1 13q14.1 Underexpression 20 [136,137] CCND1 11q13 Overexpression [ ] CCNE 19q13.1 Overexpression 20 [141,142] MDM2 12q14.3 Overexpression [143,144] p16/ink4 9p21 Underexpression 30 [ ] p27/kip1 12p13 Underexpression [ ] p21/cip1 6p21.2 Underexpression nr [151,152] Apoptosis, senescence BCL2 18q21.3 Overexpression [ ] BAX 19q13.3 Underexpression 30 [156] MYC 8q24 Overexpression [96,157] htert 5p15.33 Overexpression [158] Adhesion molecules Proteases Angiogenesis Cathepsin D 11p15.5 Overexpression 20 [159,160] E-Cadherin 16q22.1 Underexpression [105,161,162] upa/pai1 Overexpression [163,164] and PAI2 MMPs Overexpression [ ] VEGF Repair genes BRCA1 6p12 17q21 Overexpression Underexpression nr [168,169] [ ] BRCA2 13q12 13 Overexpression 20 [104] a The inactivation of TP53 stabilizes the protein, which leads to an immunohistochemical overexpression. nr: non-reported frequency., variable frequency according to the adopted positivity threshold. NB, several genes of this table present more or less defined multiple functions. breast cancers, particularly EGFR [127,128] and ERBB2, in 20 40% of cases. Only ERBB2 is amplified in 20% of invasive breast cancers [119,129,170], in 40% of comedocarcinomas [171,172], and in other cancers particularly ovarian cancer [119]. The specific ligands of ERBB2 are unknown. ERBB2 functions as a heterodimer with one of the three other members of the family, and is activated by their ligands (NDF, TGF, EGF, heregulin, and amphiregulin) [173]. ERBB genes overexpression disturbs the transducing signals and deregulates the cell proliferation: binding to the lig- and activates the tyrosine-kinase activity of the homo-or heterodimerized receptor, leads to the phosphorylation of tyrosines and the recruitment of second messengers such as phospholipase C, ras and raf (Fig. 3). ERBB2 overexpression in breast cancer cell lines increases invasion properties and metastatic capacity of the transfected cells [174,175]. The role of ERBB2 in breast tumorigenesis has been confirmed in a murine model developing breast tumors when the ERBB2 locus is amplified in an endogenous way [176]. Recent studies have enlightened new mechanisms of regulation of ERBB2, that could be used to target the gene in breast cancer treatment [177,178]. CCND1: this gene, localized in 11q13, encodes cyclin D1 [179]. The 11q13 band was first found to be frequently amplified in breast cancer [59,180]. The first two genes mapping 11q13, FGF3 (int2) and FGF4 (hst) are unlikely candidates as their expression is generally undetectable [132,181]. Cyclins activate the kinase activity of cdks (cyclins dependent kinases) by complexing with one of them. These cdks activate a number of proteins necessary for the progression of the cell cycle (Fig. 4). The increased expression of these cyclins or cdks would be tumorigenic by the deregulation of cell division. Among cyclins A, B1, C, D1, D2, D3 and E, cyclin D1 is the most frequently overexpressed in breast cancer, in 40 50% of cases [ ]. Cyclin D1 regulates the G1-S transition and RB1 phosphorylation [182] in association with cdk4 or cdk6. The amplification of CCND1 is present in only 20% of cases, which suggests other mechanisms of gene activation [101,140,183]. The overexpression of CCND1 has been correlated with the presence of estrogen receptor [142], and it has been suggested that CCND1 could reflect the functionality of ER. An overexpression of CCND1 targeted in mammary tissue of transgenic mice leads to the development of mammary tumors, which confirms the importance of this gene in the genesis of breast cancer [184]. MYC: MYC (localized to 8q24) is the only member of the MYC family (MYCN andmycl) altered in breast cancer. MYC encodes a transcription factor involved in cell growth, differentiation and apoptosis (for review, [185]). MYC is amplified and overexpressed in about 15 20% of breast tumors, this frequency varying greatly from one study to another [47,96,186]. Few cases of rearrangements (5%), and overexpression without amplification of the gene have been reported [47, ]. MYC could be involved in early stages of breast carcinogenesis [152]. MYC transgenic mice develop mammary tumors after a long latency period, suggesting that additional molecular events are probably necessary for mammary tumorigenesis [190].

7 F. Lerebours, R. Lidereau / Critical Reiews in Oncology/Hematology 44 (2002) FGFs and their receptors: fibroblastic growth factors and their receptors are a family of genes (there are ten or so FGFs and four receptors) involved in the transformation by the deregulated activity of the tyrosine-kinase receptor, following an autocrine mechanism. FGF1 and FGF2 are highly angiogenic. In breast cancer, FGF3 and FGF4, localized in 11q13, are frequently coamplified with CCND1, but much more rarely expressed than cyclind1. These two genes, initially called int2 and hst at the time of their identification at the insertion site of MMTV, are involved in the development of mammary carcinogenesis in mice infected by this virus [181]. FGFR1 is amplified in 10% of breast cancers [191]. Among the four known receptors and eight FGFs studied, only FGFR1 and FGFR4 were overexpressed in respectively 20 and 30% of breast cancers [132]. Finally, despite convincing properties for a role in breast tumorigenesis, the genetic alterations or anomalies of the FGFs and/or receptors expression are rare. Other activated genes: Other genes are frequently activated and overexpressed in breast cancer, without genetic mechanisms of these alterations being elucidated. One assumption is that the transcriptional regulation of these genes explains their abnormal expression in breast tumors. Several arguments such as the transforming potency in vitro or in vivo, or the prognostic role of the anomaly allow asserting the importance of these gene alterations in breast carcinogenesis: BCL2/BAX: the BCL2 gene localized in 18q21.3 plays an essential role in the negative control of apoptosis: it s activation contributes to the tumor development by inhibiting the cell death and this gene is usually classified in oncogenes (for review, [192]). On the opposite, the counterpart of BCL2 gene, BAX, is proaptotic ; the expression ratio of these two genes is a critical step in the induction of apoptosis. BCL2 is constantly expressed in the normal breast tissue, in about 30 45% of breast cancers, and this expression decreases with tumor progression [153,155,193]. The loss of expression of BCL2 is not only correlated with a high apoptotic index but also with high tumor proliferation rate [194]. The regulation of BCL2 expression in breast tissue could in fact be hormone dependant, as shows the increase of this expression in Estrogen Receptor positive tumors [153,156]. This data could explain the better prognosis of breast tumors that do express this antiapoptic gene [154,195]. The genetic mechanisms underlying the BCL2/BAX expression variations are unknown. CCNE: the cyclin E associates with cdk2 in the end of G1-phase, and plays as much an important role as the complex CyclinD1/cdk4 for the G1-S transition, and the phosphorylation status of RB1 [196]. In fact, Fig. 3. Signal transduction pathways (1, 2, 3, 4). (H, hormone; R, specific receptor; RTK, tyrosine kinase receptor; PLC, phospholipase C; PIP2, phosphatidyl inositol diphosphate; PI3, inositol triphosphate; DAG, diacylglycerol; PKC/PKA, protein kinase C/A; GAP, GTPase activating protein; MAPK/MEK, MAP kinase/kinase.)

8 128 F. Lerebours, R. Lidereau / Critical Reiews in Oncology/Hematology 44 (2002) Fig. 4. Cell cycle. Small arrows, activation; lines, inhibition. cyclin E is able to replace cyclin D [197]. The CCNE alterations in breast cancer have been less studied than those of cyclind1. Yet, transgenic mice overexpressing cycline, develop mammary tumors as in the case of cyclin D1 [198]. The amplification of the gene at 19q12 is rare, less than 2%, but its overexpression is more frequent, 20 30% of breast cancers [141,142]. Moreover, CCNE overexpression (associated or not to the loss of expression of p27, the regulating protein of the cyclin E/cdk2 complex) has been correlated to a less favorable prognosis, confirming the likely role of cyclin E in breast tumorigenesis [199]. As in the case of BCL2, the genetic alterations of CCNE are ignored. htert: telomerase is a ribonucleoprotein composed of different components. The telomerase insures the replication of the distal extremities of the chromosomes named telomeres. The telomere shortening is physiological along cell replication, and results in a chromosomal instability and senescence [200]. The telomerasic activity is repressed in normal tissues, and activated in germ and malignant cells [201]. This has been shown in various types of cancers including breast, and the telomerase activity overexpression in those cancers has been sometimes correlated to an unfavorable prognosis [202]. However, these results are controversial. The discrepancies between the results could be due to the dosage method of telomerase activity. The gene htert encodes the telomerase catalytic subunit. Telomerase activity seems to correlate with the restricted expression pattern of the htert gene [203]. A quantitative realtime RT-PCR study of 134 invasive primary breast cancers has shown that a high level of htert expression is correlated to histopathological factors of poorer prognosis [158]. The role of htert in tumorigenesis has been confirmed, as it has been possible to convert normal cells into malignant one by the ectopic htert expression in combination with an oncogenic allele of H-RAS and SV40-T [204]. H-ras: unlike numerous human cancers, genes of the RAS family are rarely involved in breast cancers: HRAS is only mutated in less than 10% of cases [205] Inactiated genes, tumor suppressor genes Tumor suppressor genes act in a recessive way since the appearance of the tumor seems to require the complete loss of the gene expression. This loss of expression results of a double inactivation of the two copies of the gene fitting the Knudson s model. It is generally true that cancer susceptibility TSGs are inactivated in sporadic tumors either of the same histological type or not. On the opposite, several genes inactivated in human cancers have not yet been implied in hereditary cancers Inactiation mechanisms LOH and point mutation: in the majority of cases, the inactivation of the tumor suppressor function of a gene brings a point mutation of one copy and the deletion of the remaining one. In hereditary cancers, one copy is mutated in the germ cells and the second one is lost in the tumor DNA.

9 F. Lerebours, R. Lidereau / Critical Reiews in Oncology/Hematology 44 (2002) Homozygous deletion: is the result of two independant deletions of both copies of the gene. Haplo-insufficiency: the two-hit Knudson s model is however not systematic, and the inactivation of only one copy of a tumor suppressor gene could be enough to promote tumorigenesis. Three mechanisms of this so-called haplo-insufficiency are possible: A loss of the suppressive function on downstream targeted oncogenes: this is the case for p27/kip: a reduced expression level of p27 is expected to increase the cdk/cyclin activity [206]. A dominant negative effect: the mutation of one of the copies is sufficient to suppress the wild-type activity of the gene. This mechanism is known for TP53 [207]. A transcriptional silencing of the wild-type allele: the methylation of promotor sequences rich in cytosins (CpG islands) in the 5 portion of the genes, inhibit their transcription. It is now well known that this mechanism explains a loss or a reduced expression of a TSG whereas no mutation of the gene has been identified. In particular, it has been shown for BRCA1, p16/ink4a and CDH1 in sporadic breast cancers [145,208,209] Main tumor suppressor genes implicated in breast cancers TP53: thetp53 tumor suppressor gene, located at 17p13.1, encodes a factor of transcription implicated at many levels, in the cell cycle, in the repair process, the genome stability and the apoptosis [26,210,211]. This gene has been found frequently mutated in many human cancers [212]. Germ-line mutations are responsible for Li-Fraumeni syndrome, associated with breast cancers (1% of all breast cancers), sarcomas, brain tumors and leukemias [213,214]. TP53 mutations are found in 15 35% of sporadic breast cancers (for review, [215,216]), with a higher frequency in medullar cancers, in BRCA1 linked tumors [217,218], and in aggressive or advanced tumors [219]. TP53 mutations in breast cancers are spontaneous, that is to say non induced by an identified carcinogen [216]. The gene region in 17p13 is deleted in about 50% of tumors [69,72,112,220]. But the correlation between LOH and the presence of a mutation is not systematic, suggesting either the inactivation of another gene on the same portion of the chromosome, or a dominant negative effect [112,220]. LOH at 17p13 could precede mutation occurrence [221,222]. The transfection of a wild-type p53 inhibits breast cancer cells growth, confirming its role of tumor suppressor gene [111]. The loss of the wild-type function of the gene draws a stabilization of the protein detectable by immuno-histochemistry. Nevertheless, the positivity of the immuno-histochemistry and the presence of a mutation are not always correlated [135,220]. RB1: the retinoblastoma gene located at 13q14.1 regulates the cell cycle. The dephosphorylation of its product p110 stops the cell in G1phase, and its phosphorylation draws the discharge of the E2F transcription factors and the progression of the cycle (Fig. 4). RB1 is a tumor suppressor gene: (1) it is doubly inactivated in hereditary and sporadic retinoblastoma [223]; (2) The reintroduction of the gene reverts the tumor phenotype of breast cancer cell lines that have lost the p110 RB1 protein [103]. RB1 alterations have been described in breast cancers: rare rearrangements [136,224], and a loss of expression in about 20% of cases [136,137]. Moreover, deletions of the 13q14.1 region are found in many human cancers, including 25 35% of breast cancers [61 63,225]. But the deletions have not always been correlated with the loss of expression of p110 in immunohistochemistry [225], suggesting the inactivation of another closely located gene. The search for point mutations of the remaining allele of the gene in breast cancer tumor DNA is complicated by the large size of the RB1 locus. In a series of 130 breast cancers, a reduction of RB1 expression at the RNA level has been correlated with LOH at the RB1 locus. These results question those obtained by immunohistochemistry and reinforce the assumption of the role of RB1 in at least some breast cancers (Bièche I, personal communication). BRCA1, BRCA2: these two genes have been cloned respectively in 1994 and 1995 following linkage analysis in familial breast cancer [226,227]. Unlike most known tumor suppressor genes, the identification of BRCA1 and BRCA2 mutations in sporadic breast cancers is rare [ ]. It is however possible that the alterations of these two genes in sporadic tumors are not detected by common methods of molecular biology. Indeed, large size rearrangements (e.g. deletions of one or more exons) have been found in inherited breast cancers [233]. The role of BRCA1 and BRCA2 in breast cancer genesis, including in sporadic one, is highly suggested by many arguments: 1. Both are tumor suppressor genes: they are able to revert the tumor phenotype [117] and doubly inactivated in hereditary breast tumors [234,235]. 2. A decrease of BRCA1 expression has been reported in sporadic tumors, particularly by hyper-methylation [113,115,208]. No hypermethylation of BRCA2 has been reported [236]. It seems that BRCA2 could be overexpressed in about 20% of breast cancers and this overexpression is correlated with pathological factors of poor prognosis [104]. 3. The respective loci of these two genes at 17q22 and 13q12 q13 are frequently deleted in sporadic tumors (about 35%), although it remains difficult to

10 130 F. Lerebours, R. Lidereau / Critical Reiews in Oncology/Hematology 44 (2002) exclude that the loss of another gene located in the vicinity may be critical [63,72,237]. ATM: the gene was cloned in 1995 [238]. There are several reasons to the interest given to this gene in breast cancer. Mutations of ATM are responsible for ataxia-telangiectasia, an autosomal recessive disorder characterized by cerebellar ataxia, telangiectases, immune defects, sensitivity to ionizing radiations, and a predisposition to malignancy, including breast cancers. The breast cancer risk of heterozygotes for ATM may be 5-fold greater than in the general population [239] even if these results are debated [240]. The roles of this gene in the repair process, the genome stability, and in the cell cycle upstream of TP53 gene, make it a good candidate in tumorigenesis [241,242]. In sporadic breast cancer, the region of 11q22 23 gene is deleted in 40 60% of cases [52,55 57]. The search for remaining allele mutations has been unsuccessful, and it is possible that another candidate gene is mutated in this region, or that the techniques employed have not been able to detect these mutations. Some authors have found a decrease of the gene expression [99,100] in breast tumors, others haven t confirmed this result [243]. CDH1: the CDH1 gene encodes the E-Cadherin [244]. The E-Cadherin is an adhesion molecule whose implication in metastatis has been much-studied and not much-studied [245,246]. Germ-line mutations of CDH1 have been found in familial gastric cancers [247], and this gene seems to act as a tumor suppressor gene in these cancers. The gene region, 16q22.1 is one of the most frequently lost in breast cancers [64 67]. CDH1 is mutated in about 20 40% of lobular breast carcinomas associated with a loss of the wild-type allele, and CDH1 thus behaves as a tumor suppressor in this histological type [106,248,249]. No mutation has been found in invasive ductal carcinomas, but a decreased expression of CDH1 is often observed, which is inversely correlated with the histological grade [105,161,250,251]. This reduced expression may be due partly to the hypermethylation of the promoter sequences of the gene [209]. The molecular mechanisms of tumor progression seem to differ between ductal and lobular cancers: the progression of ductal cancers could be more sensitive to the loss of only one copy of the gene, and to the reduced expression that follows. A decreased expression of CDH1 in breast cancer could be associated to a poorer prognosis [107,162]. Cdkis: the cdki are cell cycle regulators that inhibit the complex cyclin/cdk. Two families of cdki have been described: the KIP (kinase inhibitor protein) family (p21, p27/kip1, p57) and the INK4 (inhibitor of cdk4) family composed of p15, p16, p18, p19. Some of these genes work as tumor suppressor genes, as p16/ink4a (Fig. 4). p27/kip1 may play important negative regulatory roles in G1 and G0 cells. Rare mutations of p27 have been identified in breast cancers, sometimes correlated to the LOH at the locus [252,253]. While p27 is expressed at high levels in normal breast tissue, a reduced expression is observed in breast carcinomas, and this loss of expression seems to be a factor of unfavorable prognosis [148,149,199]. p16/ink4a/mts1 and p21/ CIP1/WAF (Table 2) are implicated by a reduction of their expression in numerous cancers, including a variable percentage of breast cancers, but unlike p27/kip1, their prognostic value is controversial. PTEN: the germ-line mutation of PTEN gene, a tumor suppressor gene localized in 10q23, is responsible of several diseases including Cowden disease, which is associated with a higher risk of breast cancer [254]. However, the somatic mutations of this gene in sporadic breast cancers are rare [255,256] Other genes ESR1: the Estrogen Receptor gene is located in 6q25.2 q27, a region frequently lost in the tumor DNA of breast cancers [19,37 41]. The inactivation of ESR1 could therefore favor breast tumorigenesis, and more specially explain the resistance to endocrine therapies. A reduction of the expression of the gene at the RNA level has been found in breast tumors notably by CpG methylation [130,131]. ESR mutations and abnormal transcripts have been described (for review, see [257,258]). Studies are contradictory as far as the pathogenic role of these variants in breast carcinogenesis [259,260]. Moreover, number of these variants has been also found in normal breast tissue, so that the physiological or pathological value of these abnormal RNA is uncertain [261,262]. One has no formal proof of the ESR s mutation role in the induction of a resistance to anti-estrogen [259,260,263]. Finally, the pathological value of the genetic alterations of the estrogen receptor 1 still has to be proven. IGFs and their receptors: The IGF family of growth factors 1 and 2 exercise their role in the proliferation and cell survival via IGF1R, a tyrosine-kinase receptor. The overexpression of IGF1R cooperates in the transformation induced by viruses or oncogenes. The role of IGF-1 and IGF-2 and their receptors IGF1R and IGF2R in the development and progression in breast cancer is supported by several arguments: 1. IGF-1 stimulates proliferation of breast cancer cell lines, and promotes cell migration in some breast cancer cells. Moreover, some IGFBPs (IGF binding

11 F. Lerebours, R. Lidereau / Critical Reiews in Oncology/Hematology 44 (2002) proteins) have been shown to have a role in inhibiting metastasis. 2. IGF1R is over-expressed in about 30% of breast cancers, while the locus in 15q26 is amplified in only 5% of cases [133,134,264] 3. Cells frequently overexpressing IGF2 infiltrate the breast cancer stroma. 4. The IGF2R gene has been shown to be mutated in several breast cancers [90]. The IGF2R locus in 6q25.3 is deleted in at least 30% of breast cancers, and IGF2R behaves like a tumor suppressor gene in this cancer [265]. 4. Function of implied genes In fine, it is the role of cancer genes in the cell metabolism that explains that their alterations are tumorigenic. The objective is not so much to describe these alterations, than to understand the place and functioning of each product of these genes in proliferation, differentiation, cell interactions, and apoptosis Transduction pathways The interaction between a growth factor or a hormone and a specific cell receptor triggers off a stream of events transmitting the signal up to the core. There are several transduction pathways of the signal schematized in Fig. 3. All the steps of the transduction of the signal can be altered following an abnormal expression of a growth factor or a hormone and/or it s receptor, and/or mediatory or transmission regulatory intracellular proteins. In breast cancer, activating modifications and/ or over-expressions are observed for tyrosine kinases receptors ERBB2, EGFR and to a lesser extent, FGFRs and IGF1R. The TGF is a member of the EGF family and a ligand of EGFR. It is an essential growth factor to normal and tumoral breast development: its expression is found in the epithelium and in breast carcinomas. The overexpresssion of TGF in transgenic mice induces a proliferation, and hyperplasia and breast tumors, especially in cooperation with other oncogenes ([266], for review [267]). The TGF binds itself to the serine/threonine kinase TGFRs type receptors. The role of the TGF is complex: it inhibits the cell growth by negative control of the cell cycle in normal breast and in early stages of breast cancer. On the opposite, the tumor progression could be associated to a proliferating effect of TGF [268]. Unlike colorectal cancer, no genetic alteration has been found in breast cancers for the genes of this transduction pathway, TGFRs and SMADs downstream genes. Intracellular RAS and SRC family genes are rarely altered Cell cycle The cell cycle leads to the division of the mother cell into two daughter cells passing by the phases: G1, S (replication/dna synthesis), G2 and M (mitosis). These steps require the phosphorylation/dephosphorylation of a whole series of regulating proteins, like RB1. These phosphorylations are carried out by the cdks (cyclin dependant kinases), which associates to a regulation subunit, the cyclin, in order to be activated (Fig. 4). There exist several cyclins and cdks, generating different complexes, which each have a precise role in the cycle. This is regulated by inhibitors, called cdki, which fix themselves to these complexes and inhibit the kinase activity: p16ink4a, p15ink4b, p18, p19/arf, p21/waf/cip1, p27/kip1, and p57. TP53 operates in the cell cycle by regulating the level of expression of p21, which in turn inhibits the activity of cdk2/cyclin complex. The normal control of the cell growth requires a balance between the cdk/cyclin complexes and their inhibitors. The overexpression of the cyclins or the cdks might lead to an increased cell proliferation whereas cdki might rather be considered as tumor suppressor genes. This has been shown for p16/ink4a/mts1. In breast cancer, an overexpression of Cyclins D and E is observed in respectively 40 and 20% of cases. Inversely, TP53, p16/ink4a, p27 and p21 are inactivated in a number of breast cancers (see Table 2). Finally, the majority of breast cancers present an alteration of one or several proteins involved in G1/S transition [150]. The product of MDM2, which controls negatively the function of normal TP53, is overexpressed in about 30% of breast cancers Apoptosis, senescence The apoptosis or programmed cell death is a physiological process, which importance in the neoplastic transformation and resistance to chemotherapy is well established. Apoptosis is induced by various stimuli including growth factors, DNA damages, and p53 overexpression (Fig. 5). Apoptosis is under the control of proteins localized in the membranes of cellular organites such as mitochondrions, proteins of the BCL2 gene family. Some of these proteins inhibit, others induce the apoptosis. BCL2, which inhibits the apoptosis in numerous cell types, is expressed in about 40% of breast cancers. MYC is a nuclear intermediary of signals coming from other cells controlling the start of mitosis or apoptosis. In case of DNA damage, TP53 blocks the cell cycle in G1 to enable the cell repair. If the DNA is not repaired or if the cell does not receive an appropriate differenciation signal, TP53 induces apoptosis. The senescence is the cell proliferation arrest, which precedes apoptosis. One of the molecular determinants of senescence is telomerase. In breast cancer as in a num-

12 132 F. Lerebours, R. Lidereau / Critical Reiews in Oncology/Hematology 44 (2002) ber of other cancers, an aberrant telomerasic activity (e.g. by over- or ectopic expression of htert gene) seems a crucial step in transformation Repair The instability of the tumor genome is one of first established characteristics underlying the neoplastic process. DNA synthesis errors or damages may be repaired by several means, including a proofreading activity of the polymerase, MMR genes and excisionrepair genes (involved in Xeroderma Pigmentosum and others syndromes). A failure of these repair systems leads to an increased genomic instability which may favor in turn the accumulation of others alterations. The alteration of MMR and excision-repair genes is rarely involved in breast carcinogenesis. However, TP53, ATM, BRCA1 and BRCA2, have a more or less direct role in the repair processes, in particular through the cell cycle regulation for TP53 and ATM. The role of BRCA1 and BRCA2 in the genomic stability is probable: their products join themselves respectively with hrad50 and hrad51, which are required in mitotic and meiotic recombination and in the doublestrand break repair [269,270]. In BRCA2 inactivated mouse fibroblasts, anomalies of chromatides exchange during mitosis are observed resulting in numerous chromosomal rearrangements [271] Tumor dissemination The cancer cell could be defined by its singular capacity to metastase. This complex phenomenon calls for a sequence of steps: loss of intercellular adhesion, release and migration of tumor cells, angiogenesis, adhesion in target organs (respectively to endothelial cells to the basal membrane). It is probable that all of the molecular determinants of metastasis are still not identified. However, the alteration of the expression or of the structure of some of them has been shown to contribute to the tumor progression. The loss of expression of the Cadherins, in particular E-cadherin seems to promote tumor progression by a loss of cell adhesion. The degradation of the extracellular matrix stroma, basal and vascular membranes is due to the action of 3 families of proteases, produced by the tumoral or stromal cells: upa and tpa, which are inhibited by PAI1 and PAI2; the cathepsines B, D & G; and the metalloproteases (MMPs), inhibited by the TIMPs. Among the angiogenic growth factors, one will keep in mind the VEGF, sometimes synthesized in excess by the epithelial or stromal cells in breast cancers [168,169,272]. 5. Is there a sequence of the genetic events? Tumorigenesis can be schematically divided into three steps: The initiation requires a single and irreversible deleterious genomic abnormality. The best example of it is the presence of a germ-line mutation of BRCA1 or BRCA2. It is presumed to favor the occurrence of new genetic alterations. The promotion brings several successive or simultaneous genetic and epigenetic alterations leading to the emergence of transformed cell clones. This clonal expansion results frequently from mitogenic stimuli Fig. 5. The control of apotosis. Bold arrows indicate the cells with an altered DNA (Under F. Wallet and P.-M. Martin).

13 F. Lerebours, R. Lidereau / Critical Reiews in Oncology/Hematology 44 (2002) (through growth factors and hormones), anomalies of the cell mediations and immunity, rather than from a direct alteration of the DNA. Some of these changes seem reversible. The progression consists of a series of genomic alterations, cause and consequence of an increased chromosomal and molecular instability. It leads to the emergence of more and more aggressive clones and subclones. The clinical result is the transition between in situ to invasive and metastatic carcinomas. In breast pathology, hyperplasia atypical or not and in situ carcinoma are putative precancerous histological lesions. The prominence of genetic alterations common to these lesions and to invasive carcinoma, is an argument in that line. Most genetic alterations observed in invasive breast tumors are also found, sometimes with a different frequency, in in situ cacinomas [44, ]. However, there is no clear relation between these lesions. Unlike colorectal cancer, the sequence of genetic events in breast cancer progression is not well known. This lack of knowledge is partly due to the shortage of genetic studies on precancerous lesions, and even more so on local and metastatic recurrences. Nevertheless, it is highly probable that a single genetic model can not account for breast tumorigenesis, taking into consideration the extreme geographical, clinical, histological and biological diversity of breast cancers. None of the amplified oncogenes in breast cancers is amplified in benign mastopathy [16,172,277,278]. In atypical or not hyperplasia, there is a low level of amplification of 11q13 (about 10%) [279]. The amplification would then be a late event since it appears in in situ carcinomas (for review, [280]). CCND1 is amplified in 20 30% of DCIS [139,276,279,281], ERBB2 in 10% of those of low grade and 30 60% of those of high grade [172,222,282]. These frequencies vary from one histological type to another: ERBB2 is more frequently amplified in comedocarcinomas than in other types of DCIS. Amplifications are rare in lobular cancers, either in situ or invasive. In metastasis, the amplification frequencies of MYC, CCND1 and ERBB2 are lower to those of primary tumors, suggesting their role in the genesis of the primary tumor more than in the tumor progression [157,283]. Conversely, LOH seem to occur in early lesions. Deletions have been reported in hyperplasia, and they are more frequent in case of atypy [221,277,284,285]. In DCIS, losses on 1p, 6q, 8p, 11q, 16q, 17p, 17q and 18q are reported with a frequency of at least 20% (against more that 40% for those same chromosome arms in invasive forms) [44,222,273,275,276]. The existence of LOH common to primary tumors and metastasis suggests, in the lost regions, the existence of precociously inactivated genes. Several chromosomal regions are more frequently lost in metastasis than in primary tumors, suggesting that they contain genes implied in the tumor progression: 1p35, 3p21.3, 16q22 q23, 7q31, 15q [17,286,287]. p53 is overexpressed in 10% of low grade DCIS, and in 40% of high grade DCIS, a frequency somewhat higher to that of invasive breast cancers [274]. 6. Applications for diagnosis, prognosis and treatments 6.1. Hereditary breast cancer The main application of the recent progresses in molecular genetics of cancer resulted in the knowledge of susceptibility cancer genes. Germ-line mutations of susceptibility genes allow a risk estimation of cancer. Concerning breast cancer, two high-penetrance dominant genes have been identified, BRCA1 and BRCA2, that account for the majority of inherited cases. These two genes are routinely screened for mutations in case of high-risk breast cancer history Prognostic markers Routine pathology factors have partly failed to predict the clinical outcome of breast cancer patients. The main molecular alterations likely to be of a prognostic interest in breast cancers are amplification and/or overexpression of ERBB2, CCND1 and MYC, TP53 mutations, overexpression of EGFR, BCL2/BAX, CCNE proteins, and the loss of expression of p27. Among the invasion factors, VEGF, upa and its inhibitors PAI1 and PAI2 may be useful as well. This list is not exhaustive. But very few of the numerous proposed prognostic markers are currently in wide use. In fact, the prognostic value of a molecular marker is frequently difficult to ascertain because of inconsistent or even contradictory findings from one study to another. The example of ERBB2 is revealing: after 10 or so years, and 300 or so studies, the prognostic role of this gene in breast cancer is still debated. Molecular markers have to be studied in a standardized, reproducible and quantitative way. More important, reliable conclusions about a marker will arise from large, carefully designed, possibly collaborative, confirmatory phase III studies Predictie markers A predictive factor is associated with relative benefit (sensitivity or resistance) from a specific therapy, hormono- or chemotherapy in breast cancer. Among the above-mentioned factors, some like TP53, ERBB2 and the apoptosis genes BCL2/BAX are putative predictive