Mutations in breast cancer

CNKER LETTERS Cancer Letters 90 (1995) 51-56

Mutations in breast cancer Craig S. Cropp Laboratory

qf Tumor

Immunology

and Biology,

Nalional

Cancer

Institute,

Bethesda,

MD

20892-1402,

USA

Accepted 20 December 1994

AbstFact The genetics of spontaneous breast cancer is reviewed. We have identified three regions of amplification and nine chromosomal arms with deletions in the genome. The significance and interrelations of these mutations is discussed with respect to the complex genetics of breast carcinoma. Recent work identifying a commonly deleted region between D17S846 and D17S746 is presented, which is approximately OS-l.0 Mb centromeric to the newly described BRCAl gene candidate. Possible explanations for the different locations of our deleted region and the BRCAl gene are

presented ki)Wordr:

Breasts; Cancer; Tumor suppressor genes; Deletions; BRCA 1

Breast cancer is the most commonly diagnosed cancer and the second most common cause of cancer deaths for women of the United States. It is estimated that the risk of breast cancer for American women is one in nine. Although the cause of breast cancer is not known, several risk factors seem to increase women’s risk for the disease.Some of these risk factors include menstrual and reproductive history, family/genetic history, benign breast disease, exposure to ionizing radiation, long term treatment with estrogens,and life style, especially diet [ 11. We have been studying the molecular geneticsof sporadic breast cancer. One way to understand the etiologic association between the risk factors for breast cancer and the genetic mutations of breast cancer is to view the risk factors as providing a

favorable environment in the mammary gland epithelium in which the clonal outgrowth of selected cells with somatic mutations can occur. Such mutations might then contribute to the development of neoplasia by the deregulation of normal mammary gland development. Alternatively, the mutations might provide atypical or malignant cells with a selective growth advantage, ability to metastasize,or evade host immunosurveillance. The vast majority of our breast cancer samples are invasive intraductal carcinomas. The breast tumors we use are considered ‘sporadic’ since they are collected without regard to tumor stage or grade, or patient’s history except that there has been no prior therapy. DNA is also made from normal tissue obtained from each patient, which servesas an internal control for our analysis of the

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C.S. Cropp / Cancer Letters 90 (1995) 51-56

tumor DNA. Since approximately 5%-10% of breast cancer is believed to be due to familial genetics,it is likely that a similar percentageof our samples also contain a predisposing element in their germ cells. Thus, the only commonality in our samplesare specificity of tissue and cell type. The two most commonly recognized types of mutations in the molecular genetics of solid cancers are amplification and deletion [2]. Amplification of specific regions of the cellular genomeis a dominant mutation, and is associated with the activation or over-expression of the target genewithin the amplification unit. In our studies, amplification of the following three protooncogeneshave been detected: MYC, FGF3, and ERBB2. On chromosome 8q, MYC was amplified in 32% of the breast tumors [3,4]. The second affected chromosomal region, located on chromosome llq13, was amplified in 16% of the tumors [5-71. This region contains the FGF3 (int-2) and FGF4 (hst) proto-oncogeneswhich are membersof the libroblast growth factor (FGF) gene family. In mouse mammary tumors, the mouse mammary tumor virus (MMTV) frequently activates these genes by insertional mutagenesis [I]. The third region of amplification was the ERBB2 protooncogene on chromosome 17q. This gene is a member of the epidermal growth factor receptor gene family, and is the human homologue of the rat neu oncogene. It was amplified 10% in our studies [8]. Deletions, as defined by loss of heterozygosity (LOH), are the most common somatic mutation in malignant solid tumors. In practice, LOH is identified when the tumor is missing a DNA fragment which is present in normal tissue of the same patient. Historically, such DNA fragments were generatedby restriction endonucleasedigestion to produce restriction fragment length polymorphisms (RFLPs) depending upon whether or not a single restriction site was present. A helpful development in the usefulness of RFLPs was the understanding of variable number of tandem repeat (VNTR) units [9]. In a VNTR unit, a ‘cassette’of DNA sequenceis repeated in tandem a variable number of times. The VNTR units are also known as mini-satellites. Thus, the usefulness

and information of VNTR RFLPs is significantly greater than single allele RFLPs. With the widespread availability of the polymerase chain reaction (PCR), the analysis of VNTR units has taken yet another big step in usefulnessand information content. With PCR it is now possible to quickly amplify even smaller repeat sequences, micro-satellites, from small amounts of starting DNA material [lo, 111.Generation of these PCRbasedpolymorphisms, also called sequencetagged sites (STS), has been a major research effort of many genome centers in the past few years. In primary human breast cancers, LOH is the most frequently occurring somatic mutation [ 121. The deletions that we recognize as LOH might occur as a result of interstitial deletions, chromosome loss, or aberrant mitotic recombinational events. Such mutations are believed to reveal the presenceof a ‘tumor suppressor’ gene(s)within the corresponding region of the affected homologous chromosome. The first detected tumor suppressor genewas the retinoblastoma gene (RB-1) which is now considered the paradigm. According to Knudson’s hypothesis, tumor suppressor genes contributed to neoplasia by two independent mutational events which inactivate both alleles of the gene [13,14]. One allele is lost as a result of LOH, and the other allele contains either a small deletion or point mutation which inactivates the geneproduct. In our panel of breast tumors, LOH has frequently been detected on chromosomal arms lp 1151,lq [161,3~ 1171,7q U81, 11~ [192X 13q PI, 17p, 17q, and 18q [21,22]. It is likely that multiple genetic mutations act in concert to produce a complex disease such as breast carcinoma with its metastatic potential. In agreementwith our understanding of the complex genetic interactions of malignancy, we have identified 11 pairs of mutations in primary breast carcinomas which occur together at a statistically significant frequency [2, 211. Further analysis of these pairs of mutations identified two subsetsof tumors. One subset frequently contains LOH on chromosomes1lp, 17p and 18q, whereasthe other subset frequently contains LOH on chromosomes 1p, 13qand 17q. Similar types of interacting mutations have been detected in other studies of carcinomas of the breast, colon and lung. These

C. S. Cropp / Cancer

findings that collaborating mutations may be selectedduring tumor development are another confirmation of the complexity of cancer genetics. In order to identify the actual gene that is the target of our observed LOH, a more detailed deletion map was needed.Therefore, we chose the long arm of chromosome 17, and surveyed it with 18 polymorphic markers [23]. Three independent regions of interstitial .deletions within chromosome 17qwere identified. A proximal region of deletions was defined between the loci D17873 and NME 1. This is an approximately 22 CM region and encompassesthe region which Hall et al. (241 had identified as containing the gene of hereditary breast cancer, BRCA 1. A central region of LOH on chromosome 17q is bordered by D17S86 and Dl7S21, and is estimated to be 28 CM in length. The third region of deletions is bordered by the D17S20 and D17S77loci which ti estimated to be 11CM in size. It is also possible that there is a fourth more distal region of deletions near Dl7S24 (RMU3). However, it was not possible to clearly distinguish the third region from this possible more telomeric region in our tumor samples. Our findings of distinctly separate deletions within one chromosomal arm is corroborated by the published researchof other laboratories. While suchcomplex patterns of deletions might seemsurprising, it is entirely consistent with the complex biologic changes a cell must undergo to become malignant and metastasize.It is also possible that the geneticsof breast carcinoma may be especially complex if the many predisposing risk factors are indicative of several alternative pathways to the development and progression of this malignancy. We decided to pursue the significance of LOH in our proximal region of deletions on 17q for the following reasons:(1) a high frequency of LOH at this locus, (2) Hall et al. [24] had identified this region as containing BRCA 1, the gene of hereditary breast cancer, and (3) NM23, a potential tumor suppressorgenecandidate, is located in this region. There are actually two NM23 genes, now known as NMEl and NME2, and both map to chromosome 17q21 [25-281. NM23 was discovered by Dr. Patricia Steegin the Laboratory of Pathology at the National Cancer institute by subtraction cDNA screeningof two murine melanoma

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cell lines of different metastatic potential [29]. This genewas the twenty-third nonmetastatic clone she analyzed. The gene is evolutionarily conserved, and homologous to the developmentally regulated Drosophila abnormal wing disc (AWD) gene [30]. Transfection of NMEl cDNA into the metastatic human breast carcinoma cell line MDA-MB-435 results in a 50%-90% reduction in metastasesin vivo [31], and a reduced colonization and motility in vitro [32]. Also, NMEl transfected cells exhibit features of differentiation such as the formation of ductal structures, production of basement membrane, and synthesis of sialomucin when cultured in a media containing basement membrane components [33]. Previously, it had been shown that loss of NME RNA or protein expression was signiftcantly associated with increased metastases and/or decreasedpatient survival in patients with breast cancer [34-391. Therefore, we wanted to know if loss of NMEl expression occurred in the same48 patients where we had observed LOH of NMEl, and if point mutations within the coding region of the gene occurred in the patients with LOH of NMEl . The NMEl protein was assayed in formalinfixed, paraffin-embedded sections of each tumor by immunocytohistochemistry with monoclonal antibody 301 (MAb301). This antibody is specific for the NMEl protein, and does not cross react with the homologous NME2 protein. The sections were evaluated independently by two pathologists who were blind to the genotype and patient survival data. Three classifications of NMEl staining were used by the pathologist: (1) diffuse high (DH) staining of virtually all the tumor cells, (2) focal low (FL) or heterogeneous staining when there were at least two areas of low staining similar to that of the acellular stroma, and (3) diffuse low (DL) staining where the staining intensity of the overwhelming majority of tumor cells was similar to the low staining acellular stroma. This classification permitted a quantification of what is a spectrum of biologic behavior. Of the original 48 patient tumors, 37 had informative genotypes and could be used for statistical analysis between NMEl LOH and protein expression. While a trend was observed between NMEl LOH and diffuse low NMEl expression (P <

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C. S. Cropp / Cancer Letters 90 (1995) 51-56

0.04, Armitage’s test of proportions), exceptions were noted. For example, some tumors were DL staining without NMEl LOH, and other tumors had DH staining with NMEl LOH. To determine whether NMEl LOH and/or NMEl protein expression were associated with patient survival, a logrank analysis of patient disease-free survival was conducted. An association between low (DL and FL) NMEl protein expression and shortened disease-free survival was clearly evident (P < 0.018). However, LOH of NMEl was not associated with disease-free survival. To determine if NMEl LOH was associatedwith point mutations of the remaining allele, we performed DNA sequencingof the coding region and single-strand conformation polymorphism (SSCP) analysis. However, no evidence of mutations within the coding regions was found. Our data regarding NMEl in breast cancer may be summarized as follows: (1) low NMEl protein expression is significantly associated with poor survival, confirming the results of others, (2) NMEl LOH is not associatedwith poor survival, but a trend is noted between diffuse low NMEl protein expression and NMEl LOH, and (3) no evidenceof mutations in the NMEl coding region [40]. Therefore, it is likely that mechanismsother than LOH are responsible for the reductions in NMEl expression. Possible examples of such mechanismsinclude tram- or c&acting mutations that decreasegene transcription or RNA stability. Also, unlinked trans-acting mutations might affect NMEl protein stability. Since mutations were not found in the coding region of the remaining NMEl allele, it did not fulfill the definition of a classical tumor suppressor gene.Therefore, we continued to analyze our proximal region of deletion (i.e. Dl7S73 to NMEl) for candidate tumor suppressorgeneswhich might fulfill Knudson’s hypothesis. A sub-region of this interval, between Dl7S250 proximally and Dl7S579 distally, was analyzed by PCR with seventeen polymorphic STS markers [41]. This analysis identified a smallest commonly deleted region between Dl7S846 and Dl7S746 which is estimated to be 120-150 Kb. Simultaneously, using a combination of yeast artificial chromosome (YAC) and Pl phage

clones,we in collaboration with the laboratories of Ray White and Bruce Ponder have created a physical map of the region [42]. During the past several months, the boundaries of BRCAl have continued to be refined to a smaller and smaller interval on the basis of meiotic recombination events in BRCAl linked families. The smallest interval defined for BRCAl was between Dl7S776 and Dl7378. The recent cloning of the BRCAl geneby Skolnick and colleagues [43] confirms its presence within this interval near the Dl7S855 loci. How then do we explain our commonly deleted interval which is approximately 0.5-1.0 Mb centromeric to the known BRCAl gene? There are several possible explanations. One possibility is that if the genomic size of BRCAl was very large, our commonly deleted interval might overlap with BRCAl . Although the genomic size of BRCAl is relatively large at about 100 Kb, it is not large enough to make this a likely explanation. Another possibility is that we have identified a fragile site with perhaps no biologic significance. A third possibility is that there may be more than one tumor suppressor gene in the area with relevance to breast carcinoma. At least two observations support this third possibility. First, with the exception of one paper by Bowcock et al. [44], all of the recombinational mapping studies were done with families having hereditary breast and/or ovarian cancer as opposed to families having only hereditary breast cancer without the ovarian component. Thus, there might be two closely linked genes which are independently mutated. Second, our defined interval may contain a gene more relevant or only relevant to sporadic breast cancer than to hereditary breast cancer. Support for this possibility is the lack of BRCAl mutations in the first report of 44 spontaneous breast carcinomas [45]. Accordingly, we are continuing to pursue candidate genes in the deleted interval we have identified. Defining the genetic pathways of breast cancer continues to be a substantial challenge. However, progressis being made toward a better understanding of this complex disease.As more STS markers are developed, better genetic maps will be made, and isolation of relevant genes will occur more quickly. With the cloning and characteriza-

i S. Cropp/ Canrer Letters 90 (1995 ) .
of better

health cart! for women.

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situ and invasive ductal human breast carcinoma. J. Natl. Cancer Inst., 85, 727-731. [39] Tokunga, Y., Urano, T., Furukawa, K., Kondo, H., Kanematsu, T. and Shiku, H. (1993)Reduced expression of nm23-HI, but not of nm23-H2, is concordant with the frequency of lymph-node metastasis of human breast cancer. Int. J. Cancer, 55, 66-71. [40] Cropp, C.S., Lidereau, R., Leone, A., Liscia, D., Cappa, A.P.M., Campbell, G., Barker, E., Doussal, V.L., Steeg, P.S. and Callahan, R. (1994) NMEI protein expression and loss of heterozygosity mutations in primary human breast tumors. J. Natl. Cancer Inst., 80, 200-204. 1411 Cropp, C.S., Nevanlinna, H.A., Pyrhonen, S., Stenman, U.-H., Salmikangas, P., Albertsen, H., White, R. and Callahan, R. (1994)Evidence for involvement of BRCAI in sporadic breast carcinomas. Cancer Res., 54, 2548-2551. [42] Albertsen, H.M., Smith, S.A., Mazoyer, S., Fujimoto, E., Stevens,J., Williams, B., Rodriguez, P., Cropp, C.S., Slijepcevic, P., Carlson, M., Robertson, M., Bradley, P., Lawrence, E., Harrington, T., Sheng, Z.M., Hoopes, R., Stemberg, N., Brothman, A., Callahan, R., Ponder, B.A.J. and White, R. (1994) A physical map and candidate genes in the BRCAI region on chromosome 17ql2-21. Nat. Genet., 7, 472-479. 1431 Miki, Y., Swensen, J., Shattuck-Eidens, D., Futreal, P.A., Harshman, K., Tavitigian, S., Liu, Q., Cochran, C., Bennett, L.M., Ding, W., Bell, R., Rosenthal, J., Hussey, C., Tran, T., McClure, M., Frye, C., Hattier, T., Phelps, R., Haugen-Strano, A., Katcher, H., Yakumo, K., Gholami, Z., Shaffer, D., Stone, S., Bayer, S., Wray, C., Bogden, R., Dayananth, P., Ward, J., Tonin, P., Narod, S., Brisstow, P.K., Norris, F.H., Helvering, L., Morrison, P., Rosteck, R., Lai, M., Barrett, J.C., Lewis, C., Neuhausen, S., Cannon-Albright, L., Goldgar, D., Wiseman, R., Kamb, A. and Skolnick, M.H. (1994) A strong candidate for the breast and ovarian cancer susceptibility gene BRCAI. Science,266, 66-71. [44] Bowcock, A.M., Anderson, L.A., Friedman, L.S., Black, D.M., Osborne-Lawrence, S., Rowell, SE., Hall, J.M., Solomon, E. and King, M.-C. (1993) THRAI and Dl7Sl83 flank an interval of <4 CM for the breastovarian cancergene (BRCAI) on chromosome 17q21. Cancer Res., 52, 718-722. [45] Futreal, P.A., Liu, Q., Shattuck-Eidens, D., Cochran, C., Harshman, K., Tavitigian, S., Bennett, L.M., HaugenStrano, A., Swensen, J., Miki, Y., Eddington, K., McClure, M., Frye, C., Weaver-Feldhaus, J., Ding, W., Gholami, Z., Soderkvist, P., Terry, L., Jhanwar, S., Berchuck, A., Iglehart, J.D., Marks, J., Ballinger, D.G., Barrett, J.C., Skolnick, M.H., Kamb, A. and Wiseman, R. (1994) BRCAI mutations in primary breast and ovarian carcinomas. Science,266, 120-122.