Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

The phosphatase and tensin homology deleted on chromosome 10 (PTEN) gene, also known as "mutated in multiple advanced cancers 1" or "transforming growth factor-- regulated and epithelial cell-enriched phosphatase," was recently identified as a tumor suppressor gene located at chromosome 10q 23.3 (1). Germline mutations of PTEN have been found to be linked to Cowden syndrome, an autosomal disorder characterized by harmartomas and increased susceptibility to breast and thyroid cancers (4). Inactivation of the PTEN gene has recently been detected in several types of human tumors, including glioblastoma, breast cancer, kidney cancer, prostate cancer, melanoma, endometrial cancer, and lung cancer (5). The exact function of the PTEN is not known. Previous studies demonstrated that the wild-type PTEN gene has a tumor suppressor activity in isolated cancer cells (11). Overexpression of the exogenous wild-type PTEN by transfection may inhibit the proliferation of glioma cell lines (11).

Adenovirus-mediated transfer of wild-type PTEN gene into glioma cells can suppress tumorigenicity in nude mice (12). Recently, it has been demonstrated that the PTEN gene may encode a cytoplasmic protein that has a protein tyrosine phosphatase domain and a domain extensively homologous to the cytoskeletal proteins, tensin and auxilin (1, 2, 11). These findings suggest that the PTEN gene may affect integrin or cytoskeletal function, and overexpression of PTEN can inhibit cell migration and the spreading and formation of focal adhesions (13). In the present study we investigated the role of PTEN in lung cancer invasion.

We previously established a cell-line model in human lung adenocarcinoma with different invasive abilities and metastatic potentials (14). By examining PTEN expression in these cell lines, we found that a homozygous deletion in exon 5 is associated with the high invasive ability of the cell line. We then used this highly invasive cell line to construct stable constitutive and inducible wild-type PTEN expression transfectants. We found that overexpression of the PTEN gene can inhibit lung cancer cell invasion. We further explored the possible cellular functions, as well as the downstream gene expression regulated by the PTEN gene, using a high-density complementary DNA (cDNA) microarray system to identify differentially expressed genes in PTEN-overexpressed cells (15). Several genes related to carcinoma invasion were identified using this technique.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Cell Lines

Human lung adenocarcinoma cell lines of different invasive and metastatic capacities, CL_1-0, CL_1-1, and CL_1-5 (14), were grown in RPMI #1640 medium with 10% fetal bovine serum (FBS) at 37°C, 20% O₂, and 5% CO₂. CL_1-0 is the parent cell line; CL_1-1 and CL_1-5 are sublines selected from CL_1-0 by in vitro selection with polycarbonate membrane coating with Matrigel (Collaborative Biomedical, Becton Dickinson, Bedford, MA) in a Transwell invasion chamber (14). CL_1-5 has the highest invasive ability, CL_1-1 has invasive ability in between those of CL_1-0 and CL_1-5, and CL_1-0 has the lowest invasive ability.

Reverse Transcriptase/Polymerase Chain Reaction Analysis and DNA Sequencing

Total RNA from the cell lines and transfectant cells was isolated by the single-step acid guanidinium thiocyanate-phenol-chloroform extraction method as previously described by Chomczynski and Sacchi (16). The RNA was reverse transcribed using murine leukemia virus reverse transcriptase (RT) (RNA PCR kit; Perkin-Elmer, Emeryville, CA), random hexamer, and other kit reagents, followed by polymerase chain reaction (PCR). A cDNA encoding the entire human PTEN open reading frame (1,200 base pairs [bp]) was amplified from the placenta library by PCR using sense primer 5'-GGA TCC GAC ATG ACA GCC ATC ATC AAA G-3' and antisense primer 5'-CTC GAG TCA GAC TTT TGT AAT TTG TGA ATG CTG-3'. The PTEN 5' primer (5'-ATG ACA GCC ATC ATC AAA GAG-3') and PTEN 3' primer (5'-TCA GAC TTT TGR AAT TTG TGT ATG-3') were used for analysis of PTEN expression in pIND PTEN transfectants. The primers for RT-PCR amplification for examining PTEN expression of the pCEP4 transfectant were 5' primer 5'-ATG ACA GCC ATC ATC AAA GAG-3' and 3' primer 5'-GTG GTTT TGT CCA AAC TCA TC-3'. The PCR was performed with a total of 25 cycles of 94°C for 1 min, 55°C for 1 min, and then 72°C for 3 min, and final extension for 10 min at 72°C. The PCR products were then cut from agarose gels, purified, and directly sequenced using an automated sequencing system (ABI 373A; Perkin-Elmer). Finally, the sequences were analyzed and compared with the wild-type PTEN sequence with DNAStar software (DNAStar Inc., Madison, WI).

Plasmid Construction and Transfection

An ecdysone expression system using pIND and pCEP4 plasmids (Invitrogen, Carlsbad, CA) was selected to establish stable PTEN transfectants (13). pIND PTEN and pCEP4 PTEN were created by inserting the full-length human PTEN cDNA between the HindIII and BamHI sites of pIND and pCEP4 plasmids, respectively. All contracts were sequenced entirely to eliminate PCR- generated artifacts. The CL_1-5 cells with homozygous deletion of exon 5 were selected for transfection study. The transfection experiment was carried out with pIND and pCEP4 plasmids containing the full-length PTEN cDNA insert or with plasmids containing no insert. For constitutive PTEN expression transfectants, 1 × 10⁶ CL_1-5 cells were plated in RPMI #1640 and 10% FBS and grown to 50% confluency. The cells were then transfected with pCEP4-based expression vector, with plasmid containing either the full-length PTEN insert or vector alone and lipofectin (GIBCO BRL, Gaithersburg, MD). Hygromycin B was added to a concentration of 250 µg/ml for selection of stable constitutive PTEN-expressing transfectant.

A pVg RXR vector was selected to isolate the inducible transfectants (13). The pVg RXR is a vector that expresses both ecdysone receptor and retinoid X receptor (RXR), is driven by cytomegalovirus and Rous sarcoma virus promoters, and can be selected by zeocin antibiotics. The 1 × 10⁶ CL_1-5 cells were grown to 50% confluency in a 60-mm dish and then cotransfected with 5 µg pIND and 5 µg pVg RXR in the presence of 40 µg lipofectin. Gentamicin-G418 (400 µg/ml) and zeocin (200 µg/ml) were added for selection of transfectant. The stable transfectants were then isolated 3 wk later using a ring-cloned technique.

Immunocytochemical Study and Western Blot Analysis

A mouse monoclonal antibody (mAb) (PTEN A2B1, immunoglobulin [Ig] G; Santa Cruz Biotechnology, Santa Cruz, CA), that recognizes the epitope corresponding to amino acids 388 to 400, mapping at the carboxyl terminus of the PTEN gene, was used for immunostaining and Western blot analysis to confirm the protein expression in the transfectants. The cultured cells were fixed directly with cold methanol-acetone (1:1, vol/vol) on plastic coverslips (Miles Laboratories, Naperville, IL). The fixed cells were washed with phosphate-buffered saline (PBS). After blocking with 5 mg/ml bovine serum albumin (BSA), coverslips were incubated with either mAb PTEN A2B1 (1 µg/ml) or an irrelevent mouse control antibody, followed by fluorescein-conjugated goat antimouse IgG secondary antibody (Vector Laboratories, Burlingame, CA). The cells were examined and photographed using a Zeiss Axiphot epifluoresence microscope equipped with MRC-600 laser scanning confocal imaging system (Bio-Rad, Rockville Center, NY).

For Western blot analysis, total cell lysates (50 µg of protein each lane) were separated with 12.5% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and transferred to an Immobilon membrane (Millipore, Bedford, MA). The proteins were detected with mAb PTEN A2B1, followed by the use of a biotinylated antimouse secondary antibody (Vector Laboratories). The protein bands were chromagenically stained with avidin and horseradish peroxidase (Vectastain ABC Kit; Vector Laboratories).

Cell Growth and In Vitro Invasion Assay

From each of the stable transfectants, 1 × 10⁵ cells were seeded in triplicate at each time point into six-well plates. After incubation for 24, 48, or 72 h, the cells were then trypsinized and the viable cells were counted by the trypan blue dye exclusion method using a hemocytometer.

The membrane invasion culture system (MICS) was used to measure a cell line's invasive ability (14). A polycarbonate membrane containing 10-µm pores (Nucleopore Corp., Pleasanton, CA) was coated with a mixture of laminin (50 µg/ml; Sigma Chemical Co., St. Louis, MO), type IV collagen (50 µg/ml; Sigma), and gelatin (2 mg/ml; Bio-Rad, Hercules, CA) in 10 mM glacial acetic acid solution. The membrane was placed between the upper- and lower-well plates of the MICS chamber. Cells were then resuspended in RPMI #1640 containing 10% NuSerum and seeded into the upper wells of the chamber (5 × 10⁴ cells/well). After incubation for 24 h at 37°C, cells that had invaded the coated membrane were removed from the lower wells with 1 mM ethylenediaminetetraacetic acid (EDTA) in PBS, and dot-blotted onto a 3-µm polycarbonate membrane. After fixation in methanol, blotted cells were stained with Liu stain (Handsel Technologies, Inc., Taipei, Taiwan), and the cell number in each blot was microscopically counted. Each experiment with the transfectant cell lines was repeated in triplicate.

Profiling of Gene Expression Pattern in PTEN Transfectants Using the cDNA Microarray System

To profile the gene expression patterns regulated by PTEN, messenger RNAs (mRNAs) from stable transfected cell lines with or without constitutive wild-type PTEN expression were extracted using oligotex-dT resin (Qiagen, Hilden, Germany). The quantity of 1 µg of each mRNA sample was labeled with biotin for color detection. The labeling reactions were done during reverse transcription in the presence of 6 µM random primers; 0.5 mM each of deoxyadenosine triphosphate, deoxycytidine triphosphate, and deoxyguanidine triphosphate; 40 µM deoxythymidine triphosphate; 40 µM biotin-16-deoxyuridine triphosphate (dUTP) (Boehringer Mannheim, Mannheim, Germany); 10 mM dithiothreitol; 0.5 unit/µl RNasin (GIBCO-BRL); and 200 units of MMLV RT (GIBCO-BRL) in a 50 µl solution.

A cDNA microarray system and membranes (Boehringer Mannheim) were prepared as previously described (15). The cDNA microarray membrane contains 9,600 nonredundant expressed sequence tag (EST) clones with putative gene names selected from the IMAGE consortium human cDNA libraries on the basis of the insert length and sequence of the EST clones in the Unigene database (17). These 9,600 nonredundant EST clones served as the hybridization target. The membrane carrying the double-stranded cDNA targets was prehybridized in 1 ml hybridization buffer (5× saline sodium citrate [SSC], 0.1% N-lauroylsarcosine, 0.1% SDS, 1% blocking reagent mixture (Boehringer Mannheim), and 50 µg/ml salmon sperm DNA) at 68°C for 1 h before hybridization.

After hybridization, the membrane was blocked by 1 ml of 1% blocking reagent (Boehringer Mannheim) containing 2% dextran sulfate at room temperature for 1 h and then rinsed with 1× Tris-buffered saline (TBS) buffer solution (10 mM Tris-HCl [pH 7.4], 150 mM NaCl, and 0.3% BSA). The membrane was incubated for 2 h with a 1-ml mixture containing 700× diluted STREP-GAL (1.38 U/ml, enzyme activity) (GIBCO-BRL), 4% polyethylene glycol 8000 (Sigma), and 0.3% BSA in 1× TBS buffer. The membrane was then washed with 1× TBS buffer three times for 5 min each. The color development reactions were then stopped by 1× PBS containing 20 mM EDTA.

The quantification was done by the Mu CDA program written in-house and is available via anonymous ftp at genestamp. . The program isolates differentially expressed genes by measuring the integrated density of each spot, performing regression analysis on the integrated density data, and locating the statistical outliers as differentially expressed genes.

Northern Hybridization

To confirm the results of gene detection by the cDNA microarray system, the differentially expressed cDNA clones were selected from the array membrane and the entire inserts of the clones were individually PCR-amplified to serve as probes for Northern hybridization. A 1-µg quantity of mRNA from various PTEN transfectants was isolated and poly (A⁺) mRNA was purified by two passes of total RNA over an oligo (dT)-cellulose column. Equal amounts of RNA were electrophoresed on 1.2% agarose gel in the presence of 2.2 mM formaldehyde and transblotted onto a nylon membrane. The nylon membranes were baked at 80°C in a vacuum for 2 h. The membranes were then prehybridized at 68°C in a solution containing 6× SSC, 10 mM EDTA, 5× Denhardt's solution, 0.5% SDS, and 100 µg/ml sheared salmon-sperm RNA. The PCR-amplified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNAs were [³²P]-labeled by the random prime labeling system (Amersham Pharmacia Biotech, Piscataway, NJ). The hybridization was carried out at 68°C for 24 h. The filters were washed twice in 2× SSC/0.1% SDS at room temperature for 15 min, followed by one 30-min wash in 0.1× SSC/ 0.1% SDS. The relative abundance of individual gene mRNA in each transfectant was normalized with the GAPDH mRNA band that hybridized to the [³²P]-labeled GAPDH probe.

Flow Cytometry Analysis of Integrin ₆ Expressions

The stable constitutive transfectant pCEP4 PTEN cells were subjected to indirect immunofluorescence staining for the expression of surface integrin ₆ using murine mAb against human integrin ₆ (BQ16; Acell Co., Bayport, MN). Each of the transfectant cells was grown to subconfluence and then harvested from the tissue-cultured flask with 1 mM EDTA. Cells were fixed for 30 min at 4°C in 2.5% paraformaldehyde, washed, and then blocked with 20% goat whole serum. The cells were then incubated with anti-integrin ₆ mAbs for 45 min at room temperature and washed three times with PBS. Subsequently, cells were incubated with fluorescein-conjugated goat anti-mouse IgG for 45 min at room temperature and again washed 3 times with PBS. Finally, the fluorescence intensity was analyzed by FACStar (Becton-Dickinson, Mountain View, CA). The results are expressed as mean fluorescence intensity of positive cells.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Expression of the PTEN Gene in CL₁ Cells and Transfectants

The expression of the PTEN gene in CL₁ cells with different invasive abilities was examined by RT-PCR using PTEN-specific primers and direct sequencing, as shown in Figure 1. The CL_1-0 and CL_1-1 cells expressed both wild-type PTEN and exon 5-deleted mutant alleles. The most invasive subline CL_1-5 cells expressed a PTEN mutant with homozygous deletion of exon 5. The CL_1-5 cells were then selected to construct wild-type PTEN-overexpressed transfectants. By using pIND plasmid, we established stable pIND PTEN transfectants with PTEN expression regulated by ecdysone. The pIND PTEN (+) clone contained full-length, 1,200-bp PTEN cDNA and the pIND PTEN () contained vector alone. After treatment with 1 µM muristerone A for 5 d, overexpression of wild-type PTEN could be induced. Figure 2A shows the results of RT-PCR using PTEN-specific primers, in which overexpression of wild-type PTEN gene could be induced in pIND PTEN (+) transfectant. In addition, we also established stable constitutive wild-type PTEN expression clones by using pCEP4 vector in CL_1-5 cells. Six clones were selected, namely clones 9, 10, 13, 17, 6, and 16. Clones 13, 17, 6, and 16 contained full-length PTEN cDNA, whereas clones 10 and 9 contained vector alone. Figure 2B shows the results of RT-PCR using wild-type PTEN-specific primers. Clones 13, 17, 6 and 16 expressed wild-type PTEN, whereas clones 9 and clone 10 did not. Northern hybridization was then used to examine the PTEN mRNA expression level among these PTEN transfectants, as shown in Figure 2C. A 20-fold overexpression of wild-type PTEN mRNA was observed in clones 6 and 16 as compared with the control clones (clones 9 and 10).

View larger version (11K): [in this window] [in a new window]   View larger version (34K): [in this window] [in a new window]   Figure 1.   (A) PTEN cDNA sequence of CL1-5 cells showing deletion at exon 5 from codons 85 to 164. The PTEN exon 5- deleted mutant encodes a truncated protein and the deduced amino acid sequences are compared with the wild-type PTEN. The deleted region in exon 5 is located within the putative phosphatase domain (from amino acid residue 90 to 140). (B) RT-PCR analysis of PTEN expression in each of the CL1 sublines. Specific primers were designed to detect wild-type PTEN as well as the exon 5 mutation. The RT-PCR conditions and primer sequences are detailed in MATERIALS AND METHODS. In CL1-0 and CL1-1 cells, both wild-type PTEN and exon 5-deleted PTEN were present, whereas in the CL1-5 cells, only the exon 5-deleted PTEN mutant form was detected. Wt: wild-type, delx5: exon 5 deletion.

View larger version (23K): [in this window] [in a new window]   View larger version (51K): [in this window] [in a new window]   View larger version (55K): [in this window] [in a new window]   Figure 2.   The expression of wild-type PTEN and exon 5-deleted PTEN mutant in the stable transfected cell lines were examined by RT-PCR with specific primers after transfection of the wild-type PTEN gene. In the ecdysone-inducible PTEN expression system (A), pIND PTEN (+) clone expressed a small amount of wild-type PTEN, whereas after 1 µM muristerone A treatment for 5 d, overexpression of wild-type PTEN could be induced. In the PTEN constitutive overexpression experiments using pCEP4 vectors, clones 13, 17, 6, and 16 contained full-length PTEN cDNA, whereas clones 9 and 10 contained vector alone. The RT-PCR with specific primer showed that clones 13, 17, 6, and 16 expressed wild-type PTEN, whereas clones 9 and 10 did not (B). Northern hybridization was performed to analyze wild-type PTEN expression in clones 9 and 10 (vector alone) and clones 16 and 6 (PTEN-positive) (C). Full-length cDNA, 1.2 kb, was used as a probe. Northern hybridization revealed that clones 16 and 6 expressed high levels of wild-type and mutant forms of the PTEN mRNA whereas clones 9 and 10 showed only mutant form. The transfected wild-type PTEN mRNA does not contain 3' and 5' untranslated region, which is smaller in size as compared with mutant form. Mt: mutant form, Wt: wild-type, delx5: exon 5 deletion.

Expression of PTEN Proteins in the Transfected Cells

The expression of PTEN proteins in the transfectants was investigated by a mAb PTEN A2B1, which specifically recognized the carboxyl terminus of the PTEN gene product. Figure 3 shows the immunofluorescent staining of wild-type PTEN-overexpressed cells (clone 16) and the mock transfectants (clone 10). Wild-type PTEN proteins were expressed diffusely in the cytoplasm of clone 16 cells. The clone 10 cells contained only truncated proteins which cannot be recognized by A2B1 antibody. Western blot analysis (Figure 4) also reveals the same results. Both clone 16 and clone 6 cells expressed the transfected wild-type PTEN proteins, whereas clones 9 and 10 did not.

View larger version (85K): [in this window] [in a new window]   Figure 3.   Immunofluorescent staining of wild-type PTEN-overexpressed cells (clone 16) and the mock transfectant (clone 10) by PTEN A2B1 mAb, which recognizes only the carboxyl terminus of PTEN. (A) and (C ): Phase contrast micrographs. (B) and (D): Immunofluorescent micrographs captured by confocal microscopy. A and B are mock transfectants (clone 10) which show negative in immunostaining. C and D are clone 16, which contains wild-type PTEN. The wild-type PTEN proteins are expressed diffusely in the cytoplasm of clone 16 cells. Original magnification: ×1,000.

View larger version (42K): [in this window] [in a new window]   Figure 4.   Western blot analysis of stable transfectant of wild-type PTEN-overexpressed clone (clones 16 and 6) and mock transfectants (clones 10 and 9). Clones 16 and 6 expressed wild-type PTEN proteins that can be recognized by mAb PTEN A2B1, whereas mock transfectants do not express wild-type PTEN proteins.

In Vitro Invasion Assay and Cell Growth in PTEN-Overexpressed Clones

Figure 5A shows the results of in vitro invasion assay in PTEN-overexpressed clones as compared with those of the control clones. In stable constitutive PTEN-expression transfectants, clones 6 and 16, which exhibited PTEN overexpression, showed an invasion rate that was reduced by about 50% compared with the control clones (clones 9 and 10). Cell proliferation analysis revealed that PTEN-overexpressed clones did not show any significant reduction in cell growth (Figure 6). In the ecdysone-inducible PTEN expression system, the pIND PTEN (+) cells showed a dose-dependent reduction in invasion ability after induction of PTEN expression by muristerone A (Figure 5B). These data indicate that overexpression of PTEN can inhibit invasion in CL_1-5 cells.

View larger version (38K): [in this window] [in a new window]   View larger version (33K): [in this window] [in a new window]   Figure 5.   Overexpression of PTEN can suppress the in vitro invasive ability in CL1-5 cells. In the stable constitutive PTEN-overexpressed transfectants, clones 6 and 16, which exhibited PTEN overexpression, showed an invasive ability that was about 50% reduced as compared with the control clones with vector alone (clones 9 and 10) (*P < 0.05 by Student's t test) (A). In the PTEN ecdysone-inducible expression system, the pIND PTEN was treated with muristerone A, 0.5 and 1 µM, to induce wild-type PTEN expression. The induced expression of wild-type PTEN was associated with significant reduction (*P < 0.05 as compared with noninduced control by Student's t test) of invasion ability in a dose-dependent manner (B). Each experiment was carried out in triplicate. The data are presented as means ± standard deviation (SD).

View larger version (18K): [in this window] [in a new window]   Figure 6.   The effect of PTEN overexpression on cell proliferation was analyzed in PTEN constitutively expressed clones (clones 6 and 16) and in controls (clones 9 and 10). The short-term culture showed no significant difference of growth kinetics both in the PTEN-overexpressed clones and in the mock transfectants of CL1-5 cells.

Isolation of Differentially Expressed Genes Regulated by PTEN Using the cDNA Microarray System

The stable transfectants, which constitutively overexpressed PTEN, were chosen for cDNA microarray analysis. The mRNA of each stable PTEN transfectant mRNA was isolated and then hybridized to the cDNA microarray membrane. A total of 9,600 cDNA clones in the microarray membrane were analyzed. The array signal intensities of PTEN-overexpressed clone (clone 16) were compared with intensities of the mock clone (clone 10) transfected with empty vector. As shown in Figure 7, most of the spots showed the same signal intensities, however some of the spots revealed different signal intensities. After computer analysis and normalization with control, a 2-fold difference in signal intensities was arbitrarily chosen and considered significant. An example of a close-up view shown in Figure 7 reveals that integrin ₆ and an EST cDNA clone were differentially expressed in wild-type PTEN-overexpressed cells as compared with the control clone. The array hybridization was repeated four times with the same clones on four different arrays, using a new array for each experiment. Only those cDNA clones which constantly showed at least 2-fold difference were selected for further analysis. Tables 1 and 2 show panels of putative genes that were differentially expressed in PTEN-overexpressed clones. Some of these genes were inhibited after PTEN overexpression, including laminin ₃, integrin ₆, human mRNA for Drg1, myb-related protein B, Akt2, and some EST genes. Some of the putative genes were stimulated by PTEN overexpression, including protein phosphatase 2A1B, leukocyte elastase inhibitor, nuclear factor (NF)-B, cyclic adenosine monophosphate response element binding protein (CREB), heat shock protein 90, trans-golgi network (TGN 46), DNA ligase 1, ubiquitin protease (unph), and some EST genes. Among these differentially expressed genes, integrin ₆, laminin ₃, Akt2, and protein phosphatase 2A1B have previously been reported to be associated with carcinoma invasion (18).

View larger version (80K): [in this window] [in a new window]   Figure 7.   Profiling of the differentially expressed gene pattern in stable constitutive PTEN-overexpressed clone (clone 16) and in mock transfectant (clone 10) was performed by the cDNA microarray technique. The cDNA microarray membrane contains 9,600 putative genes and EST cDNA clones. A total of 5 µg mRNA from each of the transfectants was labeled with biotin-16-dUTP by RT-PCR and hybridized to the cDNA microarray membrane, followed by color development. The image was recorded by a digitized drum scanner (A). A close-up view (B) shows that the 6 subunit of one of the cDNA clones is suppressed in the PTEN-overexpressed clone, while one EST clone is upregulated by PTEN.

Northern Hybridization Analysis of PTEN-Regulated Genes

Figure 8 shows the Northern hybridizations of representative genes differentially expressed in PTEN-overexpressed clones and mock clones. The heparin-binding epidermal growth factor (EGF)-like growth factor, RTP genes, Akt2 genes, and integrin ₆ were suppressed by PTEN overexpression, whereas ubiquitin protease (unph) and some EST genes were upregulated by PTEN overexpression.

View larger version (74K): [in this window] [in a new window]   Figure 8.   Representative PTEN-regulated genes were analyzed by Northern hybridization. To confirm the results of differentially expressed genes obtained by the cDNA microarray technique, each candidate cDNA clone was isolated and PCR-amplified, and its sequence confirmed. Northern hybridization was then performed using these representative cDNA clones as probes. A quantity of 1 µg mRNA from each of the stable constitutive PTEN transfectants was electrophoresed and transferred to nylon membrane for hybridization. Clones 6 and 16 are PTEN-overexpressed clones, whereas clones 9 and 10 are mock transfectants. Suppression of EGF-like growth factor, Akt2, integrin 6, and differentiation- related gene 1 (Drg 1), and increased expression of ubiquitin protease (unph) and one EST clone were observed. The housekeeping gene GAPDH was used as an internal control in these experiments.

Flow Cytometry Analysis of Integrin ₆ Expression

Because the cDNA microarray study showed that integrin ₆ genes are downregulated by PTEN overexpression, we carried out a flow cytometry analysis for integrin ₆ subunit protein expressions. The stable constitutive transfectants of pCEP PTEN cells were subjected to indirect immunofluoresence staining using mAb against integrin ₆. The PTEN-overexpressed clones (clones 6 and 16) showed reduced integrin ₆ expression as compared with the mock transfectants (clones 10 and 9) (Figure 9A). In the ecdysone-inducible PTEN expression system, the pIND PTEN (+) cells also showed reduction in integrin ₆ expression after induction of PTEN expression by 1 µM muristerone A (Figure 9B). The flow cytometry studies confirmed that PTEN overexpression might downregulate integrin ₆ subunit expression at the protein level.

View larger version (30K): [in this window] [in a new window]   Figure 9.   Flow cytometry analysis of integrin 6 expression by specific mAb in stable constitutive wild-type PTEN expression transfectants (clones 6 and 16) and mock transfectants (clones 9 and 10) revealed that 6 integrin expression was downregulated by PTEN (*P < 0.05 by Student's t test as compared with clone 9) (A). The flow cytometry analysis of integrin 6 expression in stable inducible PTEN expression transfectant pIND PTEN revealed that integrin 6 expression is suppressed after induction of PTEN overexpression by 1 µM muristerone A (*P < 0.05 by Student's t test) (B). Each experiment was carried out in triplicate. The data are presented as means ± SD.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The results of this study demonstrate that exon 5 deletion of PTEN is associated with a high invasive ability in our human lung cancer metastatic cell line model. Overexpression of wild-type PTEN inhibited the invasive ability of these cells. We further investigated the downstream genes regulated by PTEN expression using high-density cDNA microarray techniques. Several differentially expressed genes associated with carcinoma migration and invasion were identified which could be modulated by expression of PTEN, including integrin ₆, laminin ₃, Akt2, and protein phosphatase 2A1B. We also found a panel of genes that could be modulated by PTEN expression. The PTEN-downregulated genes included heparin-binding EGF-like growth factor, a transcription inhibitor 1d-2H, urokinase-type plasminogen-activator, Myb-related protein B, S-arrestin, and some EST clones. The PTEN-upregulated genes included leukocyte elastase inhibitor, ubiquitin protease (unph), secreted phosphoprotein 1, human canalicular multispecific organic anion transporter, Not 56-like protein, NF-B, clK3, CREB, protein phosphatase 2A1B, DNA ligase 1, trans-golgi network protein (TGN 46), glutamyl aminopeptidase A, and others. Northern hybridization and flow cytometry analysis confirmed the downregulation of integrin ₆ by PTEN both in mRNA and at the protein level. Therefore, downregulation of integrin ₆ may also be involved in the mechanisms accounting for the suppression of lung cancer cell invasion by PTEN.

Because of its high association with deletion or mutation in many primary human cancers and several familial precancerous disorders, PTEN is thought to be a tumor suppressor gene (1). However, its exact cellular function remains unclear. PTEN encodes a 47-kD protein that contains the sequence motif highly conserved in the members with protein tyrosine phosphatase activity (8). PTEN has been shown in vitro to possess phosphatase activity on phosphotyrosine and phosphoserine/threonine-containing synthetic substrate (24). Cumulative evidence has shown that PTEN may play a critical role in regulating fundamental cellular function and tumor progression. PTEN can suppress cell growth and inhibit tumorigenesis (11, 12, 25). The growth suppression of PTEN was mediated by its ability to block cell-cycle progression in the G1 phase. The cell-cycle kinase inhibitor P27^K1P1 was significantly increased and protein kinase B/Akt was inhibited after expression of PTEN (19). Loss of PTEN may result in decreased sensitivity of mutant cells to apoptotic stimuli and thereby allow them to survive and proliferate (18). Recently, PTEN has been demonstrated in vitro to dephosphorylate phosphatidylinositol 3,4,5-triphosphate, a product of phosphatidylinositol 3 kinase (PI3 kinase) (15). This is consistent with the notion that the phosphatase activity of PTEN is required for tumor suppressor activity. PTEN also has sequence similarity to tensin (1, 2), which is a cytoskeletal protein that binds to actin filaments at focal adhesions and is tyrosine-phosphorylated upon integrin-mediated cell adhesion (26). Further, recent reports have indicated that PTEN not only can suppress growth of various cells and tumorigenicity, but also is capable of regulating cell biologic activities unrelated to growth. Tamura and colleagues demonstrated that overexpression of PTEN in NIH 3T3 fibroblasts can downmodulate integrin-mediated focal adhesion formation and organization of actin containing cytoskeleton through their intrinsic phosphatase activity (13). They concluded that PTEN expression could suppress cell spreading and migration. Recently, Tamura and coworkers also demonstrated that PTEN could suppress cell invasion through dephosphorylation of focal adhesion kinase (27). These data suggest that in addition to tumor suppressor activity, PTEN may also function in the regulation of dynamic cell-surface interactions that involve integrins, focal adhesion kinase, cell migration, and the cytoskeleton. Our results further confirm that PTEN may also play an important role in tumor progression by suppression of carcinoma invasion. The invasive ability of CL_1-5 cells was significantly reduced in both the PTEN-overexpressed stable constitutive and the inducible transfectants. The reduction of invasive abilities is associated with downregulation of integrin ₆ expressions.

The exon 5 deletion is one of the hot spots for PTEN mutations. Furnari and associates previously reported two glioma cell lines with exon 5 deletion (11). The possible consequence of this deletion mutation may associate with inactivation of phosphatase activity and suppression of tumor growth (11). However, there was no detailed information on the mutation site. In our cell line system, the cell growth was not significantly reduced in exon 5-deleted cells. We examined 15 lung cancer cell lines, two of which expressed exon 5 deletion at the same sites. Both of these two cell lines (PC14 and CL_1-5) showed higher invasive ability (data not shown). Because only limited cell lines were tested, further studies are necessary before we can draw any conclusions. Northern blot analysis showed that the CL_1-0 and CL_1-1 cells contain both wild-type and mutant PTEN. Possible explanations include the presence of splicing variants (11) or the presence of heterogeneous cell population due to genetic instability in cancer cells.

The results of cDNA microarray analysis in this study indicate that several migration- and invasion-related genes were modulated by PTEN overexpression. The expression of integrin ₆ was reduced in PTEN-overexpressed clones both in mRNA and at the protein level. Other related genes, such as proto-oncogene protein kinase B/Akt, were downregulated by PTEN, whereas protein phosphatase 2A1B was upregulated by PTEN. These results are consistent with previous reports (19, 20). The integrin ₆ subunit, in combination with the ₄ subunit, may form a heterodimer ₆₄ integrin. The integrin ₆₄ is a receptor for the laminins, and plays an important role in the biology of invasive carcinoma (28). This integrin is essential for the organization and maintenance of epithelial structure (29) and may mediate the formation of stable adhesive structures, termed hemidesmosomes, that link the intermediate filament cytoskeleton with extracellular matrix (30). Persistent expression of ₆₄ integrin is present in many tumor cells that do not form stable adhesive contacts but rather exhibit the motile phenotypic characteristic of invasive carcinoma (31). A recent study indicates that ₆₄ integrin can promote carcinoma invasion through activation of PI3 kinase activity (19). The integrin ₆₄ may regulate PI3 kinase activity and further activate a downstream small G protein Rac. Our data on integrin ₆ is consistent with these previous reports (31). PTEN may play a role in the suppression of carcinoma invasion through downregulation of integrin ₆ subunit expression.

Phosphatidylinositol 3,4,5-triphosphate, a product of PI3 kinase, is a key molecule involved in the cell growth and signal transduction pathways. Recently, PTEN has been shown in vitro to dephosphorylate phosphatidylinositol 3,4,5-triphosphate (15). This evidence suggests that PTEN may negatively regulate the intracellular level of phosphatidylinositol 3,4,5-triphosphate through direct dephosphorylation (15). This process may also lead to decreased phosphorylation and activity of protein kinase B/ Akt (18). Therefore, PTEN expression may inhibit Akt expression through the reduction of phosphatidylinositol 3,4,5-triphosphate level (18). These results are consistent with the data obtained in this study by the cDNA microarray technique. Protein kinase B/Akt is a key signaling component lying downstream of PI3 kinase (20). Akt-2 is a serine-threonine kinase that can phosphorylate several proteins which could mediate cell growth, apoptosis, and tumor progression (21, 22). Both PI3 kinase and protein kinase B/Akt are involved in tumor progression and carcinoma invasion (18). In a recent study of pancreatic ductal adenocarcinoma cell lines, transfection with antisense Akt2 constructs was able to inhibit tumorigenicity and invasiveness (23). PI3 kinase activity is required for the formation of lamellae, dynamic sites of motility in carcinoma cells (19). Protein phosphatase 2A is one of the enzymes involved in the maintenance of cytoskeletal polymerization and also influences tumor invasion and metastasis (32, 33). Reduced phosphatase 2A enzyme activity has been demonstrated in metastatic cells as compared with nonmetastatic cells in Lewis lung carcinoma (34). The nonmetastatic cells may acquire the invasive and cytoskeletal characteristics of metastatic cells upon inhibition of their serine/threonine protein phosphatase (35). Our data show that protein phosphatase 2A1B is stimulated by PTEN-overexpression, indicating that protein phosphatase 2A1B may also play a role in the suppression of invasive abilities in the PTEN-overexpressed CL_1-5 cells.

The cDNA microarray analysis also revealed a panel of proto-oncogene, kinase, and transcriptional factors which can be modulated by PTEN overexpression. Some of the functions of these genes have been previously reported and may possibly be related to tumor suppressor activity by PTEN. Some of these genes have never been reported before. Thus, the results of this study demonstrate the feasibility of the cDNA microarray technique in profiling thousands of downstream gene changes regulated by the tumor suppressor gene PTEN. Our cDNA microarray and color detection systems are highly accurate and easy to use. The quantitation accuracy is comparable with conventional Northern hybridization. The standard deviation of these systems is 7% (15). We selected a cutoff value of a 2-fold difference for the differential expression of genes. Lowering the cutoff value would suggest that more genes had been modulated by PTEN. Therefore, due to the selection criteria for the cutoff valve, some of the genes affected by PTEN overexpression may not be included in Tables 1 and 2.

In conclusion, PTEN overexpression may not only affect cell proliferation and tumorigenicity but also suppress migration and carcinoma invasion in our lung cancer cell line model. Using the high-density cDNA microarray technique, we were able to analyze the downstream gene changes modulated by PTEN. Our results show that downregulation of ₆ and protein kinase B/Akt and upregulation of protein phosphatase 2A1B may also contribute to the suppression of invasive ability in PTEN-overexpressed cells. We also demonstrated the existence of a panel of genes that can be modulated by PTEN. Further studies are necessary to elucidate the exact function of these genes and their relationship to PTEN expression. The cDNA microarray technique may be useful in these investigations.