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Alterations of the PPP2R1B Gene in Human Lung and Colon Cancer

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Science  09 Oct 1998:
Vol. 282, Issue 5387, pp. 284-287
DOI: 10.1126/science.282.5387.284

Abstract

The PPP2R1B gene, which encodes the β isoform of the A subunit of the serine/threonine protein phosphatase 2A (PP2A), was identified as a putative human tumor suppressor gene. Sequencing of thePPP2R1B gene, located on human chromosome 11q22-24, revealed somatic alterations in 15% (5 out of 33) of primary lung tumors, 6% (4 out of 70) of lung tumor–derived cell lines, and 15% (2 out of 13) of primary colon tumors. One deletion mutation generated a truncated PP2A-Aβ protein that was unable to bind to the catalytic subunit of the PP2A holoenzyme. The PP2R1B gene product may suppress tumor development through its role in cell cycle regulation and cellular growth control.

Carcinomas of the lung and colon account for over 270,000 new cases of cancer each year in the United States (1). Many human cancers contain mutations in tumor suppressor genes, which often map to genomic regions that exhibit allelic loss, or loss of heterozygosity (LOH), in tumors. LOH at chromosome 11q22-24 has been associated with lung, colon, breast, cervical, head and neck, and ovarian cancers, as well as melanoma (2). Introduction of a normal chromosome 11, or a derivative t(X;11) chromosome containing 11pter-q23, can reverse the tumorigenic potential of lung (3), breast (4), and cervical carcinoma cells (5) and Wilms tumor (6) when introduced into nude mice. These studies suggest that one or more tumor suppressor genes are located centromeric to the t(X;11) breakpoint at 11q23.

To identify the tumor suppressor genes on chromosome 11 that are inactivated in lung cancer, we mapped the t(X;11) breakpoint relative to regions of frequent LOH (7) and defined a minimum critical region between D11S1394 and D11S689(Fig. 1A). This region of chromosome 11q23 demonstrated high-frequency LOH in a variety of cancers, including lung and colon tumors. Evaluation of LOH in 28 lung cancer and paired normal cell lines revealed allelic loss in 71% of the cancer cell lines. Two polymorphic DNA markers, D11S1647 andD11S1987, showed allelic loss in 42.9 and 46.2% of the cancer cell lines, respectively (8), and both markers were lost in 28.6% of the cell lines.

Figure 1

(A) Physical map of chromosome 11q23-24 showing PPP2R1B and the LOH critical region. Polymorphic markers from chromosome 11 radiation hybrid maps were used for LOH analysis (7). Expansion of the map and high-resolution mapping were done by PAC clone isolation (11). Markers PP1410, PP960, PP389, and PP1 were derived from portions of an EST sequence (GenBank accession number M65254). (B) The predicted amino acid sequence of PP2A-Aβ, determined from the sequence of PPP2R1B. Alignment of human PP2A-Aα with human PP2A-Aβ, pig PP2A-Aβ, and XenopusPP2A-Aβ. Boxed amino acids represent highly conserved residues defining 15 internal repeats (14). The sequence is separated according to repeats; numbers at left indicate the repeat number (dashed lines separate discontinuous sequence). Shaded amino acids, indicated in the human PP2A-Aβ sequence, represent mutations found in lung and colon tumors. Sequence alignment was carried out with Multiple Sequence Alignment (Infomax, Bethesda, Maryland) and MEGALIGN (DNAStar, Madison, Wisconsin). Portions of the PPP2R1Bsequence were determined previously (14) (GenBank accession number M65254). The complete sequence of the human PPP2R1Bgene has been deposited in GenBank (accession number AF087438).

On the basis of these results, the region betweenD11S1394 and D11S689, and especially that between D11S1647 and D11S1987, was systematically surveyed for candidate tumor suppressor genes. Over 100 candidate genes and expressed sequence tag (EST) markers were identified from a radiation hybrid map of human chromosome 11 (available athttp://ftp.well.ox.ac.uk) and from the Human Gene Map (available athttp://www.ncbi.nlm.nih.gov/SCIENCE96/ResTools.html). One of the EST sequences (M65254) was found to correspond to a subunit of the serine/threonine protein phosphatase 2A (PP2A). PP2A is an important regulatory enzyme that down-regulates the mitogen-activated protein kinase (MAPK) cascade, relays signals for cell proliferation, and has been linked to carcinogenesis (9). The PP2A holoenzyme exists in several trimeric forms consisting of a 36-kD core catalytic subunit, PP2A-C; a 65-kD structural/regulatory component, PP2A-A; and a variable regulatory subunit, PP2A-B, which confers distinct properties on the holoenzyme. Each subunit exists as multiple isoforms encoded by different genes, so that there are many forms of the PP2A trimer, differing in expression patterns and specificity. The gene identified at chromosome 11q23, denoted PPP2R1B according to standardized nomenclature, encodes the β isoform of the structural/regulatory A subunit PP2A-Aβ. PP2A-Aβ is necessary for interaction of the catalytic PP2A-C and variable PP2A-B subunits and is critical for phosphatase activity (10).

The precise physical location of PPP2R1B was determined by colocalizing it within P1-derived artificial chromosome (PAC) (11) clones that contained sequence-tagged sites (STSs) on chromosome 11q22-23 (Fig. 1A). PAC clone pDJ433L20 containedPPP2R1B as well as markers SHGC9837 andD11S966E and was localized to a 15 centiray region between markers D11S1647 and D11S1987 on 11q22.2 (12). Thus, the PPP2R1B gene is located in a region showing high-frequency LOH (Fig. 1A).

The complete sequence of PPP2R1B was determined by a combination of EST analysis (13), cDNA sequencing (14), and 5′ rapid amplification of cDNA ends (RACE) (15). The sequence predicts a 601–amino acid protein with extensive homology to the PP2A-Aβ subunits of pig and Xenopus and to the human PP2A-Aα isoform (Fig. 1B). Both PP2A-Aα and PP2A-Aβ are composed of 15 internal repeat sequences, each consisting of two amphipathic helices necessary for binding PP2A-B and PP2A-C (16).

To determine whether human tumors contain alterations inPPP2R1B, we amplified the coding regions by reverse transcriptase–polymerase chain reaction (RT-PCR) (17) and carried out direct DNA sequencing of samples from 130 cancer-derived cell lines and 70 primary tumors, including lung, colon, breast, and cervical tumors. Two of the tumors generated altered PPP2R1B amplification products. H1450 cells had a 1.8-kb product representing the wild-type PPP2R1B and a second 1-kb product (Fig. 2A). Sequencing of the latter revealed an internal in-frame deletion of 867 base pairs (bp), which is predicted to produce a truncated PP2A-Aβ lacking amino acids 230 to 518 (Fig. 2B). The 1.8-kb PCR product contained an A1540→G transition, which changes a highly conserved Asp504 to Gly. Thus, both alleles of PPP2R1B are altered and possibly inactivated, whereas the matched lymphoblastoid cell line from the same patient, BL7, contains no detectable alterations. In H220 cells, one of the PPP2R1B alleles had a 143-bp deletion, resulting in a frameshift and a loss of 83 amino acids at the COOH terminus of PP2A-Aβ (Table 1).

Figure 2

Mutations of thePPP2R1B gene in cancer cell lines and primary tumors. (A) RT-PCR amplification of the coding sequence ofPPP2R1B derived from lung cancer cell line H1450, normal control lung tissues CCD-8 and CCD-19, matched lymphoblastoid cell line BL7, and an unrelated lymphoblastoid cell line BL2009. PCR products were separated on an 0.8% agarose gel containing ethidium bromide. The 1.8-kb amplification product was present in all samples, and the 1-kb PCR product, representing the mutant ΔPPP2R1B containing an internal deletion, is present in lysates from H1450 cells. (B) Sequencing of PPP2R1B alterations. Nucleotides 708 to 726 indicate an 867-bp deletion in onePPP2R1B allele of cell line H1450 as compared to that of the lymphoblastoid matched control cell line BL7. (C) Nucleotides 292 to 304 show a G298→A substitution in one allele of cell line H2009 but not in the other allele. (D) Nucleotides 43 to 58 show a G51→C substitution in cell line H838 when compared to a normal tissue control sample CCD-8. A wild-type copy of the PPP2R1B transcript was not detected in cell line H838. (E) Nucleotides 1049 to 1066 show a 135-bp deletion in one allele but not in the other allele of primary lung tumor T12.

Table 1

PPP2R1B sequence alterations in lung and colon cancer; nts, nucleotides; aa, amino acid; CAC, colon adenocarcinoma; SCLC, small-cell lung carcinoma; NSCLC, non–small-cell lung carcinoma; Δ, deletion; →, nucleotide or amino acid alteration. “Wild type not detected” indicates the probable deletion of the allele through LOH.

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DNA sequencing of PCR amplification products revealed 18 alterations in PPP2R1B in 11 tumor-derived cell lines and primary tumors (Table 1). The alterations include deletions, frameshifts, and point mutations leading to nonconservative amino acid substitutions (Table 1). Cell line H2009 carries one allele with a G298→A transition (Fig. 2C), which changes a conserved Gly90 to Asp, as well as a wild-typePPP2R1B allele. The matched lymphoblastoid cell line BL2009 has both G298→A and wild-type coding sequences (18), which suggests that the lung cancer patient from which these cells are derived harbors a germline mutation in thePPP2R1B gene. Although it is possible that this alteration is a polymorphism rather than a mutation, it was not detected in 200 other normal and malignant cell lines. The alteration Gly90→Asp was found in two separate tumors and not in surrounding normal tissue, which suggests that this site may be a hot spot for mutation. One colon adenocarcinoma, T25, had two alterations in the same PPP2R1B allele (Table 1). Many of the alterations affect conserved amino acids within the repeat sequences necessary for PP2A-Aβ binding to the catalytic C subunit (16) and thus may destabilize the enzyme complex. In particular, deletions and frameshifts that alter repeats 11 to 15 (Fig. 1B) are likely to affect binding of PP2A-Aβ and enzyme activity (16).

Single nucleotide alterations were confirmed at the genomic level by sequencing of PCR-amplified genomic sequence from tumor samples T64, T68, H838, and H2009. Additional noncoding alterations were detected, including a G→T transversion in H220 in an exon/intron boundary, which presumably leads to a splicing error resulting in the loss of one exon, as well as the observed frameshift deletion. ThePPP2R1B genes from tumors T9 and T12 each carry a point mutation inside intron 8 that could also lead to splicing errors responsible for the observed deletion. None of the sequence changes identified in primary tumor genomic DNA were detected in the genomic DNA from adjacent normal tissue (19).

To determine if the sequence alterations in PPP2R1B might produce biochemical changes leading to altered PP2A activity, we performed immunoblot analysis with anti–PP2A-Aβ immunoglobulin. Lung cancer line H1450 has two PPP2R1B alterations, a deletion in one PPP2R1B allele, and a missense mutation in the other allele of PPP2R1B. Lysates of H1450 were found to contain an immunoreactive truncated PP2A-Aβ protein of 45 kD in addition to the wild-type 65-kD PP2A-A protein (Fig. 3A). This alteration might be expected to compromise a highly conserved region of the protein necessary for PP2A-Aβ to form a functional trimer with PP2A-B and PP2A-C (20). To determine whether PP2A-C binding was altered, we coimmunoprecipitated the PP2A-A, PP2A-B, and PP2A-C subunits with a polyclonal antibody against the PP2A-C subunit (Fig. 3B). H1450 lysates showed a decreased amount of PP2A-A and no 45-kD PP2A-Aβ, which suggests that both the truncated and the missense A subunits have decreased affinity for the C subunit (Fig. 3C).

Figure 3

(A) Identification of PP2A-Aβ proteins by immunoblotting. PP2A-Aβ was detected in lysates of normal lung tissue samples CCD-8 and CCD-19, cell line H1450, and BL7 lymphoblastoid control cells, with anti–PP2A-Aβ immunoglobulin (31). The 65-kD PP2A-Aβ protein was detected in all cells tested, and H1450 lysates contain an additional 45-kD band representing the mutant subunit. The 40-kD band is an actin control. (B) Coimmunoprecipitation of PP2A-A and PP2A-C subunits from H1450 cells with anti–PP2A-C immunoglobulin (32). Immunoblots of whole-cell lysates with both anti–PP2A-A and anti–PP2A-C show (lane 1) full-length PP2A-A (65 kD), truncated ΔPP2A-Aβ (45 kD), and PP2A-C (36 kD). The supernatant after immunoprecipitation with anti–PP2A-C (lane 2) contains PP2A-A, Δ PP2A-Aβ, and PP2A-C. The supernatant after washing of the immunoprecipitation pellet (lane 3) contains PP2A-A, indicating that it is dislodged from the complex during the wash. The immunoprecipitate (lane 4) contains decreased amounts of full-length PP2A-A and PP2A-C but lacks ΔPP2A-Aβ, which suggests that ΔPP2A-Aβ cannot bind PP2A-C. The control (lane 5) represents the reactivity of anti–PP2A-C immunoglobulin and horseradish peroxidase–conjugated anti-immunoglobulin in the absence of cell lysates. (C) Immunoprecipitation of PP2A-A and PP2A-C subunits from normal lung tissue with anti–PP2A-C immunoglobulin. The whole-cell lysate (lane 1) contains PP2A-A (65 kD) and PP2A-C (36 kD). The supernatant after immunoprecipitation with anti–PP2A-C (lane 2) contains PP2A-A and PP2A-C (lane 2). The supernatant after washing (lane 3) lacks PP2A-A, indicating that normal PP2A-A remains bound to PP2A-C during the wash procedure. The immunoprecipitate contains both PP2A-A and PP2A-C (lane 4). Lane 5 is a control lane with antibodies without cell lysate. The goat anti–PP2A-Aβ immunoglobulin was cross-reactive with the PP2A-Aα isoform as well as with PP2A-Aβ.

It has been postulated that protein phosphatases are involved in the suppression of cellular growth and cancer development by antagonizing protein kinases, many of which act as oncoproteins. A gene encoding a putative protein tyrosine phosphatase, PTEN/MMAC1, was recently shown to be mutated in human brain, breast, and prostate cancer (21, 22). PP2A also plays an important role in cell cycle checkpoint control (23), inhibits nuclear telomerase activity (24), and is a target for chemical tumor promoters (25), DNA tumor viruses, oncogenes, and components of cell growth control. Polyomavirus T antigen and SV40 small t antigen bind to PP2A-A, displacing PP2A-B and inhibiting PP2A phosphatase activity, interactions that are thought to mediate viral transformation (26, 27). The HOX11oncogene interacts with PP2A, inhibiting activity and disrupting a G2/M cell cycle checkpoint (28). Casein kinase 2α inhibits cellular transformation by ras and suppresses cell growth by enhancing PP2A activity (29).

Our results show that PPP2R1B alterations occur in human tumor-derived cell lines and solid tumors that display a high frequency of LOH on chromosome 11q23. Fifty-five percent (6 out of 11) of the tumors with PPP2R1B alterations have full or partial deletions of one allele, combined with deletions or missense alterations in conserved regions of the other allele, leaving the cells functionally null for PPP2R1B. The genes encoding the many isoforms of the PP2A-B subunit make up a large and diverse multigene family. At least five isoforms of the PP2A-B subunit have been discovered and map to other regions of the human genome that display frequent LOH in cancer (30). This suggests that the search for mutations in other components of the PP2A family and the characterization of the roles PP2A plays in tumor development may open new avenues for diagnosis and therapy of cancer.

  • * To whom correspondence should be addressed. E-mail: gevans{at}mcdermott.swmed.edu

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