[CANCER RESEARCH 56. 2400-2404, May 15, 1996]

p27/Kipl Mutation Found in Breast Cancer1

Konstantin S. Spirin,- Jean F. Simpson, Seisho Takeuchi, Norihiko Kawamata, Cari W. Miller,

and H. Phillip KoefflerDivision of Hematology/Oncology, Cedars-Sinai Research Institute. UCLA School of Medicine, Los Angeles, California 90048 [K. S. S., S. T., N. K., C. W. M., H. P. K./, and

Department of Pathology. City of Hope National Medical Center. Duarte. California 91010 ¡J.F. S.]

ABSTRACT

The p27/Kipl protein belongs to the recently identified family of proteins called cyclin-dependent kinase inhibitors. These proteins play animportant role as negative regulators of cell cycle-dependent kinase activity during progression of the cell cycle. Since cyclin-dependent kinase

inhibitors can inhibit cell proliferation, they may have a role as tumorsuppressor genes. To determine whether p27 alterations may be involvedin tumorigenesis, we examined its mutational status in 36 primary breastcarcinomas and 9 breast cancer cell lines using PCR-single-strand con-

formational polymorphism, direct DNA sequencing, and Southern blotanalysis. Southern blot analysis showed no homozygous deletions of thep27 gene in either the clinical samples or cell lines. Two point mutationswere found in primary tumors. One represents a previously undescribedpolymorphism at codon 142; another is a nonsense mutation at codon 104.The latter mutation was absent in the normal matched control sample,and, in addition, it was accompanied with the loss of heterozygosity (LOH)of a microsatellite marker in the vicinity of the ¡til gene on chromosome12pl3. These data indicate that ¡¡17mutations are a rare event in breastcancer, but may play an important role in the development of a minorityof these cancers. Furthermore, LOH analysis of the /_'/>/,<locus revealed

that an additional four of six matched DNA samples had LOH at 12pl3but did not have an alteration of the ¡¡27gene, suggesting that anothertumor suppressor gene is located on the short arm of human chromosome12 which may be frequently involved in the pathogenesis of breast cancers.

INTRODUCTION

Some of the cell cycle regulators have recently been implicated inoncogenic transformation of cells, including the cyclins active in G,and their respective CDKs,3 whose activities are regulated by a set

CDKIs ( 1-3). During G,, D-type cyclins form complexes with CDK4

and/or CDK6 and phosphorylate the Rb protein, which results in therelease of the E2F family of transcriptional activators, inducing entryinto the S-phase of the cell cycle (4-7). Cyclin E assembles withCDK2 and CDK3, and in collaboration with D-type cyclins, cyclinE/CDK2 and CDK3 contribute to the entry into the S-phase andregulation of early stages of DNA synthesis (8-12).

Activities of the cyclin-CDK complexes are negatively regulated by

a recently discovered and growing family of CDKIs (reviewed inRefs. 1, 3, 4, and 13-15). These proteins form two distinct classes

based on their structural and functional homologies. One class includes so-called INK4-proteins: pl6/INK4A (16), pl5/INK4B (17,

18), pl8/lNK4C (18, 19), and pl9/INK4D (19, 20). Each contains

Received 12/12/95: accepted 3/18/96.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported in part by N1H Grants CA43277. CA26038. CA427IO.CA70675-01, and DK42792. by the Concern Foundation, and by the Parker Hughes Trust.H. P. K. is a member of the UCLA Johnson Comprehensive Cancer Center and holds anendowed Mark Goodson Chair of Oncology Research at Cedars-Sinai Medical Center/

UCLA School of Medicine.2 To whom requests for reprints should be addressed, at Cedars-Sinai Medical Center.

UCLA School of Medicine. 8700 Beverly Boulevard.. D-5065. Los Angeles. CA 90048-1865. Phone: (310) 855-7758; Fax: (310) 659-9741.

3 The abbreviations used are: CDK/cdk, cyclin-dependent kinase; CDKI, cyclin-de

pendent kinase inhibitor: LOH. loss of heterozygosity; MDE. mutation detection enhancement; SSCP. single-strand conformational polymorphism; Rb. retinoblastoma gene

product.

four ankyrin repeats, forms complexes with CDK4 and/or CDK6 andthe D-type cyclins, and has a functional activity dependent on the

presence of a normal Rb protein (18, 19). Maximum expression ofpl6, pl8, and pl9 peaks in the middle of S-phase in proliferating cells

(18, 21). This is coincidental with one of the functions of INK4proteins, which is to inactivate G, CDK-cyclin complexes during theS-phase (18). Overexpression of INK4 proteins can block cells in G,

(22). Inactivation of the pl5 and pl6 proteins results in the illicit entryof the cell into the S-phase and appears to contribute to cellular

transformation, suggesting that the INK4 proteins may function astumor suppressor genes (1, 23, 24). In confirmation of this proposal,a high frequency of pi5 and p!6 gene deletions and pI6 mutationsoccur in tumors and in the cell lines of specific tissue types (1, 13).

The other class of CDKIs includes p21/WAFl (also known asMDA6, CIP 1, CAP 20, or SDH; Refs. 25-30), p27/Kipl (31-34), and

p57/Kip2 (35, 36). These proteins are structurally and functionallyunrelated to the 1NK4 genes. The p27 protein has 42% amino acidhomology (sequence similarity) with p21 and 47% similarity with thep57 protein within the N-terminal domain which mediates inhibition

of CDK. Each of them has a nuclear localization signal in theirC-terminal domains (32, 33, 35-38). In comparison to INK4 proteins,

the p21/p27/p57 family (Kip proteins) shows a wide specificity andspectrum of inhibitory activities. All of these proteins are able toinhibit kinase activities of preactivated G, cyclin E-cdk2 and cyclinD-cdk4/6, S-phase cyclin A-cdk2 as well as the mitotic cyclin B-cdc2

(25, 31, 35, 36). Overexpressed Kip proteins cause cell cycle arrest(32, 33, 37, 38). The p21 and p27 proteins are expressed in most cells,creating an inhibitory threshold in G,; in contrast, expression of thep57 protein seems to be tissue specific (31, 36, 39). Expression of p27is up-regulated by exposure of the cell to several antimitogens (31, 32,40-42). These data suggest that in vivo the Kip proteins may serve

both redundant and specialized functions, including the possibility ofbeing tumor suppressors.

Previous studies have shown alterations in the cell cycle regulatoryproteins in breast carcinomas, including overexpression and amplification of cyclin genes (43-46), inactivation and deletions of the Rb

gene (47, 48), alterations of the p53 gene or abnormal cytoplasmiclocalization of the protein product (47-50), and deletions and muta

tion of the pl6/INK4A gene in breast cancer cell lines (51, 52). Thep27 gene is located on chromosome 12pl3 (53-55). Chromosomal

deletions and translocations at this locus are frequent in the male germtumors as well as lymphoid and myeloid malignancies (56-64),

showing the presence of a potential tumor suppressor gene in this area.To determine whether p27 alterations have a role in the formation ofbreast cancer, we examined the status of the p27 gene in primarytumors and cell lines using Southern blot analysis, PCR-SSCP studies,

and DNA sequencing of aberrantly shifted bands observed usingSSCP. We also analyzed LOH of human chromosome 12p usingmicrosatellite markers vicinal to p27/Kipl in a limited number ofmatched samples.

MATERIALS AND METHODS

Samples. Thirty-six primary breast carcinomas were investigated. Twenty-

seven samples were obtained from the City of Hope Medical Center, and nine

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P27/KIP1 COKI GENE IN BREAST CARCINOMAS

including seven matched normal control samples were obtained via the National Cancer Institute Cooperative Human Tissue Network. All primary tumorsamples were either grade 2 or 3 ductal invasive adenocarcinomas. Nine breastcancer cell lines were studied: SK-BR-3, BT-20, MDA-MB-436, ZR-75-1,

MB-231, BT-474, MDA-MB-468, MCF7, and BT-549. All cell lines were

obtained from American Tissue Culture Collection (Rockville. MD). The cellswere cultured according to the American Tissue Culture Collection recom

mendation.Primers, PCR-SSCP, Sequencing, and LOH Analysis. Two sets of prim

ers were used to amplify exon 1: AA2-SS2 set yielding a 276-bp fragment andAA3-SS3 resulting in the amplification of a 291-bp fragment. This primer setamplified a 5' and 3' part of exon 1, respectively (exon la and exon Ib).

Primers A7 and SS4 were used to amplify a 170-bp fragment that included

exon 2 and short flanking sequences. The sequences of primers were: AA2,5'-TAGAACTCGGGCAAGCTGCCCTTCT-3'; SS2, 5'-ACCCGGGAGAA-AGATGTCAAACGTG-3'; AA3, 5'-ATTCTATGGTTGGGGAAGGGT-3';SS3, 5'-AGTACGAGTGGCAAGAGGTG-3'; A7, 5'-GTTTACGTTTGAC-GTCTTCTGAG-3'; and SS4, 5'-GTTTTTTCTAATAAAGATTGTGTGTTC-3'.

The PCR conditions for exons la and Ib were 30 cycles of 94°Cfor 30 s,60°Cfor 1 min, 72°Cfor 30 s and for exon 2 were 34 cycles of 94°Cfor 30

s, 56°Cfor 1 min, and 72°Cfor 30 s. Each 30-ml reaction contained 100 ng

DNA, 0.2 M primers, 200 mM deoxynucleotide triphosphates, and 0.5 ml Taqpolymerase (Life Technologies, Inc., Gaithersburg, MD). Magnesium (1.5mM) was used for amplification of both exons, and 2 mCi [a-32P]dCTP (3000

Ci/mmol; NEN/Dupont, Boston, MA) were added to each reaction. The PCRproducts were diluted 10 times in loading buffer containing 96% formamide,denatured by heating at 95°Cfor 5 min, and chilled on ice. Electrophoresis was

performed through a 6% polyacrylamide MDE (AT Biochem) gel with 10%glycerol at 500 V for 24 h. The gel was dried and exposed at —¿�80°Cwith

Kodak X-OMAT AR film (Kodak, Rochester, NY) and intensifying screen.

The DNA bands that migrated abnormally were excised and eluted in TEbuffer. The eluted DNA was reamplified, and sense and antisense strands weresequenced directly using a double-stranded DNA-sequencing system according to the manufacture's protocol (Life Technologies, Inc.). The samples were

electrophoresed on 7% polyacrylamide-8 M urea gels and analyzed using

autoradiography.To confirm the frequently observed polymorphism in exon Ib of p27 [base

substitution (GTC->GGC) at codon 109 (54, 55, 65), which generated a new

restriction site for Bgll], the PCR products for exon Ib were digested with Bgll(Promega; Madison, WI), separated onto a 6% nondenaturing polyacrylamidegel. and examined for the appearance of two bands.

For LOH analysis, 100 ng DNA from the tumors and their matched normalcontrol samples were amplified with primers designed to the polymorphicmicrosatellite loci DÌ2S89.DI2S98, D12S320, D12S358. and Ì2S269locatedon chromosome 12pl3, as described (58, 66). The number of cycles wasoptimized for each set of primers to achieve the best results in LOH detection.A total of 2 /xCi [a-32P]dCTP was added to each reaction. Fragments were

separated on 5% denaturing polyacrylamide-8 M urea gels and analyzed using

radioautography.Southern Blotting. For Southern blotting analysis, sufficient intact DNA

was available for 28 primary tumor samples and 9 cell lines. Five /ng DNAwere digested with 50 units fcoRI restriction enzyme (Life Technologies, Inc.)for 2 h and separated on 0.8% agarose gels in Tris-acetate buffer (73) at 35 V.

The gels were treated for 15 min in 0.25 M HCI, then twice for 15 min in 0.5N NaOH and transferred to Hybond N+ nylon membrane (Amersham, Inc.,Arlington Heights, IL) in 0.5 M NaOH. Filters were washed in 4X SSCsolution (IX SSC = 0.15 M NaCl and 0.015 M sodium citrate), air dried, and

A123456

m m < T T555

Bage t agct

l =

Cagct agct

gat-»-

g•¿�

•¿�c

T C T NFig. 1. Detection of point mutations in the p27/Kipl gene by PCR-SSCP analysis and

DNA sequencing in breast carcinomas. A. the DNAs from tumor and normal samples wereamplified with exon Ib primers and separated on 6% MDE gels. Three electrophoreticpatterns (B/B. A/B. and A/A on Lanes 1-3, respectively) were identified by PCR-SSCP ¡n

both normal and tumor samples from a variety of individuals and represents GTC (alíeleA) to GGC (alíeleß)base substitution at codon 109. Lane 4 (sample 13) and an arrowshow an aberrantly migrating band, which on sequencing (B) represents a silent mutationat codon 142; Lane 5 (sample 32) shows a slightly shifted band which represents anonsense mutation at codon 104 and its normal matched control DNA is shown on Lane6. B. p27/Kipl silent mutation (sample 13) detected by direct sequencing of DNA,amplified from shifted band, and excised from MDE gel (Lane 4, Fig. IA). Sequence ofantisense strand shows a base substitution (arrows) from guanine to adenine in the sensestrand (ACG —¿�>ACA), leading to no amino acid sequence change. C, a hemizygous

p27/KipI nonsense mutation (sample 32) detected by direct sequencing of DNA fragments, amplified from genomic DNA of a breast carcinoma sample (D and normalmatched control DNA (AO.A cytosine to thymidine (CAG —¿�>TAG, arrows) substitution

created a stop codon TAG (nonsense mutation) at position 104 in the amino acid sequence.T. breast carcinoma sample; N, normal matched DNA sample; C, normal control humanbone marrow DNA.

baked at 80°Cunder vacuum for 2 h. Prehybridization and hybridization were

performed in 5X SSC, 50% formamide, 5% dextran sulfate, 0.02% BSA,0.02% poly vinyl pyrrolidone, 0.02% Ficoll, 100 fig/ml sheared salmon spermDNA, and 1% SDS.

The blots were probed with a p27 DNA fragment which contained the 5'

portion of the coding sequence of the gene (67). The probe was generated byPCR using the SS2 and the A2 primers (5'-AGAACTGTCGGTTGCAG-GTCGCTT-3'; Ref. 65). This probe could detect an 11-kb fragment that

includes both exons of the p27 gene. A probe for myeloperoxidase was usedas control (68). Each probe was labeled with [a-32P]dCTP (3000 Ci/mmol;

NEN) using a random primer labeling system (Life Technologies, Inc.).

RESULTS

To identify potential point mutations, small deletions, and insertions oflhep27/Kipl gene, we analyzed 36 primary breast carcinomasand 9 breast cancer cell lines using PCR-SSCP. We detected two

Table 1 Analysis of the p27/Kipl gene in priman' tumors and cell lines of breast carcinomas

AlterationsSpecimenPrimary

tumorsCell

linesTotalSample36945Southern

blot0/270190/36PCR-SSCP2/360/92/45A/A22426Alíele

type"A/B12315B/B224Mutations6202

" AlíeleA represents the sequence GTC: alíeleB is GGC at codon 104 of exon 1 of the p27/Kipl gene.' Sample 32 had a CAG •¿�TAG codon 104 nonsense mutation; sample 13 contained a ACG

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•¿�ACA codon 142 silent mutation.

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P27/KIPI COKI GENE IN BREAST CARCINOMAS

31TN

33TN

34TN

35TN

*mP27

B

29 30 31 32 33 34 35TNTNTNTNTNTNTN

D12S89Fig. 2. Detection of the LOH of the human chromosome 12 at the locus 12pl3 studied

by using a polymorphism of exon I of the p27/KipI gene (A) and by a polymorphicmicrosatellile marker. DI2S89. located near the p27/KipI gene (B). A. the breast tumor (Dand normal matched control DNA (N) were amplified with p27 exon Ib primers, andfragments were separated on a 6% MDE gel. Samples 31 and 33 showed LOH of the Aalíele;sample 35 showed retention of heterozygosity. and sample 34 was noninformative.Numbers above the gels, case numbers. B, DNA from tumor (T) and normal matchedcontrol samples (N) were amplified with the primers to the polymorphic microsatellitemarker DÌ2S89,and fragments were separated on a 5% denaturing polyacrylamide gel.LOH was found for samples 29, 32. and 33, samples 30, 34. and 35 retain heterozygosity,and sample 31 was noninformalive. (Tumor sample 32 also has the nonsense mutation atcodon 104 of the p27/Kipl gene.)

alterations in the 3' part of exon 1 (exon Ib, Lane 4, sample 13; Lane5, sample 32, Fig. \A); no mutations were observed in either the 5'

part of exon 1 (exon la) or the second exon, and no hemizygousdeletions were found on the Southern blot analyses (data not shown).

Direct DNA sequencing showed that one of the mutations was a Gto A substitution (ACG —¿�»ACG) at codon 142, which represents asilent mutation with no amino acid changes (Thr —¿�»Thr; sample 13,

Fig. In). The other was a hemizygous nonsense mutation where a Cto T substitution at codon 104 created a stop codon (CAG —¿�»TAG)

instead of a glutamine (sample 32, Fig. 1C). Sequencing of a matchednormal control (Fig. 1C) revealed a wild-type p27 molecule.

A previously described polymorphism was found in exon Ib (Fig.\A; Ref. 65), where we observed three different electrophoretic patterns (A/A, A/B, and B/B) using PCR-SSCP analysis. Direct DNA

sequencing revealed that alíeleAA represents a sequence found in theGenbank entry (U10906); alíelefifi represents a base substitution atcodon 109 (GTC —¿�»GGC) that resulted in an amino acid change from

valine to glycine. Analysis of our samples showed that the B alíelewas less frequent than the A alíelein both the cell lines and clinicalsamples (Table 1).

From the seven patient samples for which we had normal matchedDNA, four were informative. LOH of the A alíelewas found in two ofthe matched samples, although no mutation of the p27 gene wasdetectable (Fig. 2A, samples 31 and 33). Therefore, LOH near the p27gene was examined using the microsatellite markers DÌ2S89,D12S98, DI2S320, D12S358, and D12S269 (Figs. 2B and 3; Refs. 58and 66). Marker D12S89 was located in close proximity to the TELIgene on chromosome 12pl3; marker DÌ2S269is near the p27 geneand markers DÌ2S98,12S320, and Ì2S358are located between theTELI and p27 genes (56, 58). Analysis revealed that five of sevensamples had LOH of at least one of the seven markers (Figs. 2B and3; Table 2). Of interest, sample 32, which had a p27 nonsensemutation and no normal alíele,also had LOH of the DI2S89 marker,further confirming that the normal p27 alíelehad been lost.

DISCUSSION

The p27/Kipl protein is a member of the CDKI family of proteinsidentified as an important regulator of the cell cycle. Recent studies

Z9 30 31 32 33 34 35

D12S89

D12S98

D12S358

D12S320

D12S269

P27

Fig. 3. Summary of LOH analysis of chromosome 12 in breast carcinomas. Sevensamples used in experiments are presented. Status of each chromosome locus is indicatedby shading as LOH (black), retention of heterozygosity (white), and not informative(cross-hatched). Top of each column, patient numbers.

Table 2 LOH analysis of the p27/Kipl gene

LOH using microsatellites on 12p 13

Case29303l32*333435LOHof/>27NI"RLLLNIRD/2589LRNILLRRD/259«LRNININILRDI2S358LNILNINIRRD/25520NIRLNILRNID/25269NINILNILRR

' NI, noninformative; L. LOH present: R, LOH absent.' Hemizygous nonsense mutation at codon 104.

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P27/KIP1 CDKI GENE IN BREAST CARCINOMAS

indicate that the p27 protein mediates growth arrest in response toextracellular growth inhibitors (31, 32, 40-42). Loss of this growth

inhibitory pathway could result in inappropriate cellular proliferation.To determine whether p27/Kipl alterations were involved in breast

cancer, we examined 36 primary breast tumors and 9 breast cancercell lines using PCR-SSCP, DNA sequencing, and Southern blot

hybridization. Two point mutations were found. One represents apreviously undescribed polymorphism in exon 1; the other is a hem-

izygous somatic nonsense mutation at codon 104. The latter mutationwas accompanied with the LOH near the p27 gene (D12S89) on the12pl3 locus. Thus, one alíeleof p27 was mutated and the other alíelewas apparently lost, suggesting that these two genetic events playedan important role in the development of the breast cancer.

The significance of this mutation is yet to be elucidated. Translationof mRNA with a stop codon at 104 position results in the expressionof a truncated protein with an intact CDKI inhibitory domain (37, 38),but loss of the C-terminal domain results in the loss of the nuclear

localization signal. Studies have shown that the related p57/Kip2protein without the C-terminal domain accumulates exclusively in the

cytoplasm (36). Furthermore, many truncated proteins have a shorthalf-life.

To our knowledge, only two other tumors have been reported thathave a p27/Kipl mutation, although over 500 tumors have beenexamined: a stop codon at position 76 was detected in one case ofadult T-cell leukemia, and hemizygous deletion of the p27 gene wasdescribed in one case of B-immunoblastic non-Hodgkin's lymphoma

(67). We previously have reported that p21AVAFl mutations are alsoextremely rare. This is surprising because p53 is an upstream activatorof p21/WAFl, and p53 is frequently mutated and becomes inert as atranscriptional activator (69, 70). Taken together, this family of CD-

KIs may provide an invaluable function for cellular viability, and mostof the mutations of this class of genes may result in cell death ratherthan transformation, but mutations that preserve some of their functional activities might play an important role in cancer development.

Cytogenetic analysis for chromosomal alterations in breast cancerhas revealed that alterations of chromosome 12 were one of the mostfrequent events in this cancer (71, 72); nevertheless, to our knowledge, a detailed allelotyping of the short arm of chromosome 12 hasnot been reported in breast carcinomas. In this study, we had onlyseven matched samples, but analysis of these samples surprisinglyshowed that five had LOH at locus 12pl3. This region has also beenfound to be altered in hematopoietic malignancies (56-58). Within

this region is the p27/Kipl and TELI loci (58); the latter can translocate and form chimeric proteins in several hematopoietic malignancies (59-64). In this study, four of the five cases with LOH had no

detectable abnormality of p27/Kipl, suggesting that another tumorsuppressor gene is located on human chromosome 12p and may befrequently altered in breast cancer.

ACKNOWLEDGMENTS

We would like to thank Gail Jao and Kelly Chung for excellent secretarialhelp.

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1996;56:2400-2404. Cancer Res   Konstantin S. Spirin, Jean F. Simpson, Seisho Takeuchi, et al.  

Mutation Found in Breast Cancerp27/Kip1

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