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Role of FHIT in Human Cancer

From the The Kimmel Cancer Center, Jefferson Medical College, Philadelphia, PA, and Istituto Nazionale dei Tumori, Milan, Italy.

Address reprint requests to Carlo M. Croce, MD, Thomas Jefferson Medical College, 233 South 10th St, BLSB, Rm 1050, Philadelphia, PA 19107-5799.

ABSTRACT

ABSTRACT: Through investigation of hemizygous and homozygous deletions in common human cancers, including lung cancer, we have cloned and characterized a gene at chromosome region 3p14.2, FHIT, that is inactivated in epithelial tumors, particularly in tumors resulting from exposure to environmental carcinogens. In some tumors, particularly those associated with environmental carcinogens, alterations in the FHIT gene occur quite early in the development of cancer. In other cancers, Fhit inactivation seems to be a later event, possibly associated with progression to more aggressive neoplasias. Thus, detection of Fhit expression by immunohistochemistry in premalignant and malignant tissues may provide important diagnostic and prognostic information.

CANCER IS A GENETIC disease resulting from multiple, sequential genetic changes affecting oncogenes, tumor suppressor genes, and modifiers. Because of this multistep process, most human malignancies show various degrees of genetic heterogeneity even if they originate from single cells. Thus, cancer cells of the same clonal tumor mass may respond differently to chemotherapy or radiation therapy. Although most of human leukemias and lymphomas carry consistent chromosomal rearrangements, predominantly chromosomal translocations or inversions that activate specific oncogenes^1,2 or cause loss of function of specific tumor suppressor genes,³ thereby initiating the process of malignant transformation, it is not known what the initiating events are for some of the most common human malignancies, the malignant epithelial tumors such as lung, breast, and prostate cancer.

If we could identify the earliest genetic changes that initiate the process of malignant transformation in solid tumors, we may be able to detect these changes in premalignant lesions and develop new drugs or novel therapeutic approaches to eliminate premalignant cells, providing new opportunities for cancer prevention. In addition, we could use the genes involved in these changes, their protein products, or the biochemical pathways in which they are involved as targets for the development of new anticancer drugs.

CLONING OF THE FHIT GENE

For these reasons, we have recently focused on the frequent genetic changes involved in common solid malignancies and their premalignant conditions. Cytogenetic analysis of lung cancers^4,5 and bronchial dysplastic lesions⁶ has indicated that the short arm of chromosome 3 is often involved in deletions in these pathologies. Thus it seemed logical to speculate that alterations in the gene(s) on the short arm of chromosome 3 (3p) may occur in preneoplastic lesions (bronchial dysplasia) and set the stage for the development of malignant tumors. Deletions of the short arm of chromosome 3 are also observed in a variety of other common tumors, such as breast cancer, head and neck cancer, gastrointestinal cancer, esophageal cancer, and cervical cancer.⁷ To identify the gene or genes on chromosome 3p that may be involved in the initial steps in the development of these tumors, it was first necessary to define the chromosomal location of these genes. In the case of leukemias and lymphomas, the chromosomal position of the genes involved in cancer initiation is often easy to define because of the consistent and frequent chromosomal translocations or inversions that initiate the process.^1,2

In the case of solid tumors, to position the critical genes, we frequently rely on studies of loss of heterozygosity (LOH), on identification of chromosomal regions that are homozygously deleted, or on the presence of rare mutations or translocations present in families that may lead to loss of function of tumor suppressor genes. Therefore, we carried out LOH studies of kidney and gastrointestinal tumors, comparing the DNA of microdissected tumor tissues for the presence or loss of markers on 3p; we complemented these studies by asking whether the critical region is involved in homozygous deletions in tumor-derived cell lines and primary tumors.⁸ We found that region 3p14.2 is often involved in LOH and deletions in human tumors, and we were able to narrow the region of interest to a few hundred kilobases of DNA.⁸ We also found that the chromosomal region involved in LOH and homozygous deletion in solid tumors is extremely close to a t(3;8)(p14.2;q24) chromosome translocation point in a family with hereditary renal cell carcinoma.⁸

By constructing a cosmid contig map of the critical region of 3p14.2 and using these cosmids in exon-trapping experiments, we were able to clone an exon (exon 5) and then the entire cDNA of the critical gene at 3p14.2, designated FHIT (Fig 1).⁸ As shown in Fig 1, the 1-2 Mb FHIT gene is composed of 10 exons, of which five are protein-coding (exons 5 through 9, shaded in Fig 1); it encodes a small mRNA (1.1 kb) and a small protein (16.8 kd). Interestingly, the breakpoint at 3p14.2, involved in the t(3;8) chromosome translocation observed in the familial renal cell carcinomas, interrupts the third intron of the FHIT gene, inactivating one of the two FHIT alleles (Fig 1). It is also of interest that the most common fragile site of the human genome, FRA3B, maps within the FHIT gene.⁸ It had been speculated that fragile sites observed in the human genome correspond to chromosomal regions frequently involved in rearrangements in human cancer.⁹ Thus the observation that the FHIT locus contains the most inducible common fragile site represents the first support for such speculation. The presence of the FRA3B fragile site within FHIT suggests that the fragility of this gene may make FHIT susceptible to rearrangements induced by a variety of environmental carcinogens. It also suggests that the degree of chromosomal fragility at this site may contribute to the degree of cancer susceptibility. The observation that the degree of inducibility of the FRA3B fragile site with inhibitors of DNA synthesis, such as aphidicolin, differs in different individuals implies heterogeneity of the degree of fragility in the population, suggesting that it will be important to examine the correlation between degree of fragility and tumor susceptibility.

View larger version (15K): [in this window] [in a new window]   Fig 1. The FHIT gene at the common fragile site at chromosome band 3p14.2. The fragile region is represented by the light blue area within band p14.2. Approximate positions of fragile site landmarks, such as hybrid breaks, integrations and the kidney cancer–associated familial translocation, are shown. The FHIT gene is shown on the right, with the protein coding exons represented by black blocks.

Sequence analysis of the FHIT cDNA and its protein product revealed that the gene is homologous to a family of genes characterized by a histidine triad, two of which encode yeast diadenosine asymmetrical hydrolases, enzymes that cleave Ap₄A into adenosine triphosphate and adenosine monophosphate (AMP).⁸ In experiments carried out in collaboration with Larry Barnes, we have shown that FHIT is an Ap₃A asymmetrical hydrolase that cleaves Ap₃A into adenosine 5'-diphosphate and AMP.¹⁰ This gene was named FHIT (sounds like fit), because it contains the most common Fragile site of the human genome and encodes a protein of the histidine triad family. Expression of FHIT mRNA is detectable in most tissues, and the highest levels of expression of FHIT mRNA and protein are detectable in epithelial cells and epithelial tissues.^8,11

FHIT IS INVOLVED IN DELETIONS IN HUMAN CANCER

Because the FHIT gene is large (> 1 Mb), we initially studied its structure in cancer cells by amplification and sequencing of the FHIT cDNAs and comparing these amplified cDNAs to cDNAs from normal cells. The results of this analysis indicated frequent alterations of FHIT transcripts in a variety of human cancers, including lung,¹² colon, esophageal,⁸ breast,¹³ head and neck,¹⁴ stomach,⁸ cervical,¹⁵ and pancreatic¹⁶ carcinomas. The alterations consisted predominantly of the absence of exons from the cDNAs of cancer cells.^8,12 The absence of exons in the FHIT mRNAs observed in tumors was often associated with the presence of genomic deletions within the FHIT locus in tumor-derived cell lines and tumor tissues. Thus, for cases that were examined in detail, the alterations in FHIT expression in tumor-derived cell lines and human tumors were the result of genomic changes affecting both alleles of the FHIT gene^11,17,18 (Fig 2). Similar biallelic deletions have been defined in several lung cancer cell lines (Druck et al, manuscript submitted for publication).

View larger version (11K): [in this window] [in a new window]   Fig 2. FHIT gene deletions in cancer cells. FHIT exons and the translocation position are shown by the top line. Apparent homozygous deletions were delineated using numerous oligonucleotide primer pairs to polymerase chain reaction (PCR)–amplify fragments spanning the locus. For cancer-derived cell lines that were studied by Southern blot analysis for retention of two exon copies or by inverse PCR and sequencing to define deletion end points, it has been possible to determine that the homozygously deleted regions represent the overlap between independent deletions of the two alleles, as shown for the Kato III and Siha cell lines, derived from gastric and cervical carcinomas, respectively.

LOH studies indicated that more than 90% of lung tumors showed LOH at the FHIT locus, and reverse transcriptase polymerase chain reaction analysis indicated that at least 80% of small-cell lung cancers and 40% of non–small-cell lung cancers (NSCLCs) have alterations in the FHIT gene.¹² Moreover, LOH at the FHIT locus was higher in lung cancers of smokers than nonsmokers,¹⁹ which indicates that FHIT may be a target of the carcinogens present in tobacco smoke. Recently, Nelson et al²⁰ observed homozygous deletion of FHIT exons in 13 of 30 primary lung cancers. These authors also observed an association of FHIT exon deletion with smoking duration and asbestos exposure, buttressing the argument that FHIT is a carcinogen target.

To facilitate the characterization of the FHIT deletions observed in human tumors and to examine the molecular basis of chromosomal fragility at 3p14.2, we decided to sequence the FRA3B region. Until recently, it was thought that the FRA3B fragile site was a small DNA region (< 100 kb). With the discovery of the FHIT gene and its genomic organization, it became clear that the FRA3B fragility involves a large DNA region, probably larger than 600 kb.²¹ We focused our attention on the epicenter of the fragile region surrounding exon 5, the first coding exon of the FHIT gene.^17,21

Sequencing of approximately 300 kb of the region surrounding exon 5 indicated that this region is low in Alu repeats and rich in lung interspersed nuclear element (LINE) 1 repeat elements over 1 kb in size.^17,22 Importantly, it allowed us to sequence many breakpoints involved in deletions in a variety of human tumor-derived cell lines, showing that in most cases the breakpoints involved LINE 1 elements larger than 1 kb and that many cancer cell deletions may result from homologous recombination between these elements.¹⁷ The sequencing of this region has not provided obvious clues to the molecular basis of chromosomal fragility at 3p14.2. The FHIT rearrangements observed in five tumor-derived cell lines are described in Fig 3. As shown in Fig 3, large deletions involve both alleles of the FHIT gene. Of interest is the observation of discontinuous deletions, such as those described in a breast cancer cell line derived from a patient who underwent radio- and chemotherapy.¹⁷

View larger version (15K): [in this window] [in a new window]   Fig 3. Cancer deletion end points within the fragile region. The 276-kb sequenced region allowed the identification of sequences involved in deletion in cancer cells. The gray arrowhead above the sequenced region represents the position of exon 5. Below the sequenced region, the deleted alleles of two colon cancer cell lines (LS180 and LoVo), a nasopharyngeal carcinoma cell line (HK1), and a breast cancer cell line (MB436) are shown. The filled arrows represent deletion end points within or near the clustered large (> 1 kb) LINE 1 (L1) elements. Open arrows mark deletion end points that were not in L1 elements.

LOSS OF Fhit PROTEIN EXPRESSION IN HUMAN CANCER

To assess the frequency and consequences of FHIT rearrangements in human tumors, we produced antiserum against the recombinant Fhit protein and used the antiserum to detect Fhit protein in tumor-derived cell lines and primary tumors. The results of this analysis indicated that tumor cell lines or tumors that exhibit altered FHIT transcripts and genomic FHIT alterations usually do not express Fhit protein or express reduced levels of Fhit, as determined by Western blotting and immunohistochemistry.^11,18,23 Figure 4 shows the anti-Fhit immunohistochemical staining of NSCLCs (Fig 4B and 4C) and a gastric adenocarcinoma (Fig 4D); the cells of the normal tissue stained strongly positive (Fig 4A), whereas the tumor cells were negative for Fhit expression (Fig 4B-4D). We have investigated the expression of Fhit in a series of primary lung tumors by immunohistochemistry. We found that nearly 100% of small-cell carcinomas of the lung did not express Fhit,²³ which is consistent with the detection of FHIT alterations in more than 80% of such tumors.¹² We then investigated a large collection (474 cases) of stage 1 NSCLCs by immunohistochemistry.²⁴ As summarized in Table 1, 73% of the tumors did not express Fhit, indicating a high frequency of loss of Fhit expression in NSCLC. Interestingly, there was a considerable difference in frequency of loss of expression in squamous cell carcinoma (87%) versus adenocarcinoma (59%). Sixty-nine percent of large-cell lung cancers also showed loss of Fhit expression. As shown in Table 1, loss of Fhit expression (negativity of staining with anti-Fhit antibodies) was high in tumors of smokers and lower in tumors of nonsmokers. Immunohistochemistry also allowed the detection of Fhit expression in precancerous lesions. The absence of Fhit was already detectable in bronchial dysplasia, a precancerous condition, and in carcinoma-in-situ, indicating that loss of Fhit is an early event in the pathogenesis of lung cancer (Table 2). Interestingly, the complete absence of Fhit protein was more common than p53 mutation in lung cancer and preneoplastic lesions and more frequent and earlier than alterations of the EGF receptor and overexpression of BCL2 (not shown).

View larger version (119K): [in this window] [in a new window]   Fig 4. Immunochemical detection of Fhit expression. (A) Normal lung tissue; note the mainly cytoplasmic brown staining of the bronchial epithelial cells and the near absence of staining of stromal cells. (B, C) NSCLCs; note the cytoplasmic brown staining in the normal bronchial epithelial cells (black arrow) and the negative staining of the cancer cells (white arrows). (D) Stomach adenocarcinoma; note Fhit expression in the normal glands of the antrum (black arrow), and negative staining of the adenocarcinoma cells (white arrows).

Others have reported that allele loss at the FHIT locus is more frequent in the bronchial epithelium, as well as neoplasias, of smokers than nonsmokers^25,26 and more frequent than allele loss at other suppressor loci. In conjunction with our observation of loss of Fhit protein in preneoplasia, the evidence suggests that loss of Fhit expression is an important indicator of carcinogen-induced damage and initiation of the multistep process that leads to lung carcinogenesis.

FHIT IS A TUMOR SUPPRESSOR GENE

The results of the investigation of human tumors indicate that the FHIT gene is altered by mutations, predominantly deletions, in human cancer and that such alterations are common in epithelial cancers, particularly those resulting from exposure to environmental carcinogens, such as lung, head and neck, esophageal, stomach, pancreatic, and cervical cancer.^8,11-15 Since both FHIT alleles are frequently altered in human cancers and since a family with hereditary cancer associated with a translocation disrupting one FHIT allele has been described, it is reasonable to consider FHIT a bona fide tumor suppressor gene.⁸ To demonstrate suppressor activity, we have transfected the human FHIT cDNA into four different tumor cell lines with homozygous deletions of the FHIT gene and then injected the Fhit-expressing transfectants into nude mice, showing that Fhit expression results in the loss of the ability to form tumors.²⁷ We have also transfected a kidney cancer cell line with a mutant FHIT gene in which the middle histidine of the histidine triad was changed to asparagine.²⁷ The mutant Fhit protein, with dramatically reduced enzymatic activity, still suppressed tumorigenicity, which indicates that the ability to cleave Ap₃A is not required for tumor suppression. More recent experiments indicate that the mutant Fhit protein binds Ap₃A as well as the wild-type protein, which suggests that the Ap₃A bound form of Fhit may be the active suppressor.²⁸

Otterson et al²⁹ expressed exogenous Fhit protein in a HeLa cell clone and found that the Fhit-expressing clone was tumorigenic in nude mice; however, the immunohistochemical detection of Fhit in the excised tumor seems to show that the majority of tumor cells have lost Fhit expression, probably as a prerequisite for growth.

Recently, we knocked out the murine Fhit gene in mouse embryonal stem cells and have obtained chimeric mice carrying the Fhit null allele. Mating of these chimeric mice with C57BL/6 mice resulted in the vertical transmission of the Fhit null allele. At present, we are investigating the progeny of the Fhit^+/- heterozygotes. These experiments will indicate whether the complete absence of Fhit protein results in the development of a spectrum of tumors in the mouse, as might be expected from the study of the genetic alterations at the FHIT locus in human tumors.

The null mice and the heterozygotes will also be extremely useful in studies of exposure to environmental carcinogens and in the dissection of the additional steps involved in carcinogenesis.

THE Fhit GENE OF DROSOPHILA MELANOGASTER LED TO THE DISCOVERY OF Nit

To assess whether loss of Fhit function may cause tumors in Drosophila, we needed to map the Drosophila Fhit gene and to determine whether this chromosomal site is deleted in some of the available mutant flies. Therefore, we cloned the Drosophila Fhit gene and mapped it to region 61A of chromosome 3 on the Drosophila salivary gland chromosomes.³⁰ Unfortunately, no flies with deletions of the Fhit locus are available. The sequence of the Fhit gene and its encoded protein revealed, however, that the fly Fhit protein has a stretch of 314 amino acids added to the amino terminus.³⁰ By taking advantage of this information, we have cloned the human and mouse homologs of the Drosophila DNA sequences encoding this 314 amino acid stretch and have discovered a gene homologous to bacterial and plant nitrilases (Nit). This gene, designated NIT1, is on human chromosome 1q21 and mouse chromosome 1 but is fused with Fhit in Drosophila. In Caenorhabditis elegans, Nit is also fused to the amino terminus of Fhit.³⁰ Thus, in Drosophila and C elegans, Nit and Fhit are chimeric, presumably with dual enzymatic activities (we have shown that the Drosophila NitFhit cleaves Ap₃A into adenosine 5'-diphosphate and AMP), whereas in the mouse and human, these two activities are encoded by two separate genes. Other proteins with multiple enzymatic activities have been discovered that are chimeric in one species and encoded by two or more different genes in different species. When this is the case, those genes are involved in the same biochemical or biosynthetic pathways. Thus, the biology is telling us that Fhit and Nit must act in concert. Interestingly, we observed coordinate expression of Nit and Fhit in different mouse and human tissues.³⁰ Thus, the identification of NIT1 may facilitate our investigation of the normal function of Fhit and the molecular basis of its involvement in carcinogenesis.

In conclusion, the positional cloning of oncogenes and tumor suppressor genes depends on our ability to define the chromosomal regions where they reside. In leukemias and lymphomas, chromosome translocations have provided information on the position of the critical cancer genes and have facilitated their cloning.^1,2 In most of the solid tumors, in which consistent chromosome translocations are unusual, different approaches are needed, including LOH and detection of homozygous deletions. We have used a combination of these approaches to isolate a specific locus from the short arm of chromosome 3 that is altered in some of the most common human cancers, including lung cancer, the most common cause of cancer death in the Western world.

By using this approach, we have cloned and characterized the FHIT gene at 3p14.2, which is frequently involved in deletions and Fhit loss in epithelial tumors, particularly in those tumors resulting from exposure to environmental carcinogens. This gene is also broken by a t(3;8) translocation in familial renal cell carcinoma in a large three-generation family. Replacement of FHIT cDNA in human cancer cell lines resulted in suppression of tumor growth, as expected for a tumor suppressor gene.²⁷ Thus, infectious recombinant vectors carrying the FHIT gene should be considered as potential gene therapeutic agents.

In some tumors, particularly those associated with exposure to environmental carcinogens, alterations in FHIT occur quite early in the development of human cancer.^18,24 In others, such as clear-cell kidney carcinoma, such alterations may occur during tumor progression.³¹ Thus, Fhit loss in bronchial tissue indicates the occurrence of genetic alterations associated with the early steps of carcinogenesis. Absence or reduction of Fhit expression in some kidney neoplasias may accompany progression toward a more aggressive form of disease.³⁰ Therefore, detection of Fhit expression by immunohistochemistry in premalignant and malignant tissues may provide important diagnostic and prognostic insights. The future challenge is to understand the physiologic role of Fhit and the consequences of its inactivation in the regulation of cell growth, differentiation, invasion, survival, and other relevant biologic process. Then we may develop new drugs and/or novel approaches to eliminate Fhit null cells. If this could be achieved, it could result not only in better treatment of Fhit-negative tumors but also in the ability to eliminate or reduce precancerous lesions, contributing to the prevention of some of the most common human cancers.

ACKNOWLEDGMENTS

We thank Dr Raffaele Baffa of the Kimmel Cancer Institute, Jefferson Medical College, for the photograph depicting Fhit expression in gastric cancer and Teresa Druck for preparation of the figures.

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Submitted July 1, 1998; accepted September 3, 1998.

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