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Keywords:

  • RAS;
  • RAB25;
  • breast cancer

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References

A novel breast cancer cell line (RAO-3) was established by transduction of the Q61L mutant RAS into human mammary epithelial cells that were immortalized with catalytic subunit of telomerase (hTERT). The cells displayed anchorage-independent growth and proliferation, and formed human mammary spindle cell carcinoma when injected into nude mice. Chromosome locus 1q22-23 was partially duplicated and inverted on one of the 3 chromosomes present in the cell line. We report here that mutations of chromosome 1q22-23 locus have resulted in the loss of RAB25 expression in the breast cancer cell line. Transduction of RAB25 into the breast cancer cell line arrests anchorage-independent growth. We have also demonstrated loss of RAB25 in human breast tumor tissue. These data suggest that loss of RAB25 might contribute to tumorigenesis of breast cancer, and RAB25 is likely to be an important factor in the development of breast cancer. RAB25 could be used as biological marker of breast cancer and provides a target for gene replacement therapy. © 2006 Wiley-Liss, Inc.

Breast cancer is the most common cancer in North American women. Many genes are considered to contribute to tumorigenesis of the mammary gland, such as RAS, BCL-2 (B-cell Leukemia-2) and NRG1 (Neuregulin1).1, 2, 3 A novel series of human breast cancer cell lines has been established through the use of defined genetic elements in an effort to better define the role of various oncogenes in a stepwise model to tumor progression.4 Human mammary epithelial cells (HMEC) from healthy individuals undergoing reduction mammoplasty were immortalized by transduction of either the catalytic subunit of telomerase (hTERT) after passage through stasis (RAO-1 cell line) or the human papilloma virus type16 (HPV16) E6/E7 genes. The RAO-1 cell line was then transduced with the Q61L mutant H-RAS gene; RAO-2 is a RAS-transduced derivative cell line of RAO-1 that does not show anchorage-independent growth in soft agar and is not tumorigenic in nude mice. RAO-3 and RAO-4 are human breast cancer cell lines that were derived from RAO-1 after RAS transduction. RAO-3 exclusively gives rise to human mammary spindle cell carcinomas when injected into nude mice. RAO-4 exclusively generates human mammary epithelial carcinoma when injected into nude mice. Previous studies suggested that a critical cytogenetic event on chromosome locus 1q23 was the last significant step to transformation in our RAS-driven model.4 We confirmed the rearrangement of chromosome 1q22-23 by FISH (fluorescence in situ hybridization) in the RAO-3 cell line. The expression of RAB25, one of the genes that are located in this region, was lost in some of the tumorigenic cell lines that we tested, including RAO-3 and RAO-4.

The RAB guanosine triphosphatases (GTPases) (RAS-related in brain)5 belong to the RAS superfamily of small GTPases. More than 60 different RAB family members have been identified in the human genome.6, 7 The human RAS family consists of 3 proto-oncogenes, H-RAS, K-RAS and N-RAS. Mutations leading to an amino acid substitution at the positions 12, 13 and 61 are the most common in neoplasms and experimentally induced animal tumors.8, 9, 10 A number of the RAB genes have been implicated as important regulators of vesicle trafficking.11 RAB25 belongs to the RAB11 subfamily, which includes 2 other members, RAB11a and RAB11b, and shows 63% homology with RAB11a protein.12 RAB11 family proteins have been shown to play an essential role in protein recycling from endosomes to the plasma membrane.13 The RAB proteins are ubiquitously expressed. Prominent expression of RAB25 has been observed throughout the gastrointestinal mucosa, with the highest expression seen in ileum and colon. High levels of expression are also present in the lung and kidney, with a very minor and variable level of expression in splenic tissue. No expression of RAB25 has been seen in the brain, heart, liver, skeletal muscle or the gastric wall.12 In our study, expression of RAB25 was also present in normal human mammary tissue and in cultured primary human mammary epithelial cells. It is an intriguing candidate gene for breast cancer, because RAB25 has a special GTP-binding site, DTAGLE, that differs from the GTP-binding site, DTAGQE, of other RAB proteins.12 More importantly, the GTP-binding site of RAB25 is homologous to the GTP-binding site of the Q61L mutant H-RAS, which leads to potent transformation phenotype.14 We examined the expression of RAB25 in RAO-3, RAO-4 and other established breast cancer cell lines. We found that the expression of RAB25 was lost in all of the breast cancer cell lines that contain a RAS point mutation, and it was also lost in some breast cancer tissues derived from human patients. After transduction of RAB25, the RAO-3 breast cancer cell line showed that the rate of cell growth was reduced and colony formation in soft agar was lost. Our data show that loss of RAB25 is associated with tumorigenesis in human mammary epithelial cells.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References

Cell culture

Primary mammary epithelial lines cell lines (HMEC4, HMEC 6, HMEC 25 and HMEC 26) were established according to standard protocol15 and maintained in DFCI-1 medium.16 All of the cell lines were grown at 37°C and 5% CO2 in DFCI-1 medium, except MDA-MB-231, MDA-MB-435s, MX-1,T-47D, MCF-7 and ZR-75-1 which were grown in DME medium with 10% FBS, and MDA-MB-468 which was grown in L15 medium with 10% FBS. Mammary epithelial cells (HMEC) were prepared by the method of Stampfer and Bartley.15 HMEC4, HMEC6, HMEC25 and HMEC26 are immortalized cell populations from different healthy donors who underwent reduction mammoplasties. HMEC4 and HMEC26 were immortalized by the transduction of the catalytic subunit of telomerase (hTERT), as previously described,17 after emergence from stasis. HMEC4 has been passaged in vitro for an extended period of time and will be referred as RAO-1 while HMEC26 is a relatively early-passage population. HMEC6 is a late-passage cell population of human mammary cells that has been immortalized by transduction with HPV16 E6/E7 genes and has been extensively passaged in vitro. HMEC25 is an early-passage cell population of human mammary epithelial cells that has also been immortalized through the transduction of HPV16 E6/E7. RAO-3 is a human mammary spindle cell carcinoma cell line that was derived from RAO-1 after RAS transduction and soft agar cloning, as previously described.4 RAO-4 is a human mammary epithelial carcinoma cell line also derived from RAO-1 after RAS transduction and soft agar cloning. RAO-2 is a RAS transduced derivative cell line of RAO-1 that is anchorage dependent and is not tumorigenic. MDA-MB-231, MDA-MB-435s, MDA-MB-468, MCF-7, T-47D and ZR-75-1 are established human breast carcinoma cell lines which were obtained from ATCC (Manassas, Virginia).

Tissues and primary mammary epithelium cells

Breast cancer tissues were obtained from tissue bank of the SIU Cancer Institute or from The Cooperative Human Tissue Network in Ohio State University. Both normal and tumor tissues were collected from each patient. Primary mammary epithelial cells were extracted from mammary tissue that was collected from mammoplasty samples, and fat and connective tissue were manually removed from each specimen. Mammary tissue was then cut into pieces and digested in digestion mix (14 U/ml hyaluronidase, 15 U/ml collagenase, 10 μg/ml insulin, 1× pen/strep, 10% FCS in DFCI-1 medium) at 37°C overnight. Cells were sieved through a sterile strainer, spun down at 1,500 rpm (300 g) for 5 min after PBS washing, and plated on 10-cm plate. Cells were fed every 2–3 days with DFCI-1 medium. Fibroblasts were removed by trypsin/EDTA solution treatment.

Fluorescent in situ hybridization

RAO-3 cell line was cultured, as described, and harvested, and chromosome spreads were made according to standard cytogenetic techniques.18 BAC clones on chromosome 1 (8D14, 165A08, 184N12, 196B07) were purchased from BACPAC Resource Center (BPRC) at Children's Hospital, Oakland Research Institute in Oakland, California. The probes were labeled with the Bio-Nick™ Labeling System (Invitrogen, Gaithersburg, MD). In situ hybridization was carried out essentially according to the manufacturer's protocol. To visualize the hybridization, the slides were counterstained with 4,6-diamidino-2-phenylindole dihydrochloride and observed with fluorescent microscopy.

Reverse transcriptase polymerase chain reaction

Total RNA was extracted from 2 × 107 cells of each cell line using RNeasy Mini kit (Qiagen Inc, Valencia, CA). DNA contamination was removed from total RNA using DNase I treatment. Reverse transcription to cDNA was performed with Superscript™ II RNase H reverse transcriptase (Invitrogen, Carlsbad, CA), primed with oligo (dT) 12–18 primer (Invitrogen, Carlsbad). Polymerase chain reaction (PCR) was performed on a GeneAmp PCR System 2700 (Applied Biosystems, Foster City, CA) using Taq DNA polymerase (Invitrogen, Carlsbad) according to the manufacturer's instructions. All reactions were performed for 35 cycles with the following parameters: 94°C, 30 sec; 58°C, 45 sec and 72°C, 45 sec. PCR primers were designed using Primer3 output software (www.broad.mit.edu) and used at a final concentration of 10 μM. Primer sequences and expected base pair PCR fragment lengths are shown in Table I.

Table I. Oligonucleotide Primers Used in RT-PCR
Primer namePrimer sequenceFragment size (bp)
β-actinF: 5′-AGAAAATCTGGCACCACACC-3′553
R: 5′-AGGAAGGAAGGCTGGAAGAG-3
RAB25 F: 5′-CTGGTGTTTGACCTAACCAA-3′532
R: 5′-GAGGTATTTGTGATAGGGCA-3′
RAB11a F: 5′-CCTGGTCCCACAGATACCAC-3′759
R: 5′-CTCAGACCTGGGAAATGGAC-3′
RAB11b F: 5′-CGGGACGACGAGTACGACTA-3′586
R: 5′-TGATGTCCACCACGTTGTTC-3′

Quantitative real-time PCR analysis

Expression of RAB25 was quantified using real-time PCR with fluorescence detection. The primer pair was designed as follows: 5′-CCCTCCTGGTGTTTGACCTA-3′ (RAB25 sense, nucleotide 476 to 495); 5′- TGGTAGAGTCCAGGGCTGAG-3′ (RAB25 antisense, nucleotide 675 to 694). Quantitative real-time PCR (qPCR) was performed using a Smart Cycler Real-Time PCR instrument (Cepheid Inc. Sunnyvale, CA) and the iQ™ SYBR Green Supermix kit (Bio-Rad, Hercules, CA). The PCR mix (26.5 μl) consisted of 1 μl cDNA, 1 μl with 10 μmol of each primer and 12.5 μl of SYBP Green mix. qPCR was performed for 45 cycles for 30 sec at 62°C and 1 min at 72°C. The expression of genes was analyzed using Smart Cycler Software version 1.2f (Cepheid Inc. Sunnyvale).

Construction of the RAB25 plasmid

Forward and reverse primers with an additional Xho1 and BamH1 restriction site were made. The following are the primers: RAB25F (Xho1): 5′CTCGAGCAACACACAGTATTGT-3′; RAB25R(BamH1): 5′-GGATCCTTTGTGATAGGGCTAGAAGA-3′. The cDNA of RAB25 was made by reverse transcriptase polymerase chain reaction (RT-PCR) from a total RNA of human normal breast epithelial cells, and then inserted to pLXSP vector between Xho1 and BamH1 cloning sites. The construction was verified by restriction enzyme digestion and sequencing.

Phoenix retroviral supernatant

Ten micrograms of pLXSP(empty vector) or RAB25/pLXSP DNA was used to prepare Phoenix Retroviral supernatant. Before transduction, Phoenix cells were treated with 25 μM chloroquine for 5 min. Then 10 μg of pLXSP or RAB25/pLXSP DNA was precipitated in 61 μl 2M CaCl2, 500 μl 2× HBS and q.s. to 1 ml with water. Five hundred microliters was added to one well of a 6-well plate and incubated at 37°C. Twenty-four hours post-transduction, medium was changed to 3 ml fresh DMEM containing 10% FCS. After cell incubation for another 24 hr, the supernatant was collected from transfected Phoenix cells, filtered through 0.22-μm filter, and stored at −80°C for further use.

Transduction of cells with retroviral supernatant and selection

The RAO-3 cell line was cultured in a 6-well plate. Polybrene (2,000×) (1 μl) and 0.5 ml of pLXSP empty vector or RAB25/pLXSP viral supernatant was added to each well and allowed to incubate for 18 hr. The cell colonies were selected by 2.5 μg/ml puromycin for 2 weeks. Expression of RAB25 in transduced cells was determined by RT-PCR.

Cell growth observation

At day zero, 5 × 104of each cell population of interest was plated on a 6-well plate. Cells were cultured in DFCI-1 medium at 37°C with 5% CO2. The number of viable cells was determined at 24, 48, 72 and 96 hr after seeding by trypan blue staining.

FTI-277 treatment

FTI-277 (farnesyltransferase inhibitor) (250 μg) (Calbiochem, San Diego, CA) was dissolved in 5.6 μl DMSO. After complete dissolution, a 10 μM solution of FTI-277 was made in DFCI-1 medium, and this served as the treatment medium. DMSO (5.6 μl) was also dissolved in same volume of DFCI-1 medium, and this served as the DMSO control medium. DFCI-1 medium was used as a general control medium. At day zero, 105of the RAO-3 cell population was plated on a 6-well plate in each kind of medium. Cells were cultured at 37°C with 5% CO2. The number of viable cells was determined at 24, 48, 72 and 96 hr after staining with trypan blue.

Soft agar assay

Six-well plates were filled with 2 ml of 0.66% noble agar in DFCI-1 medium as a bottom layer. Cells (105) were suspended in 1 ml of 0.33% noble agar in DFCI-1 medium and plated onto the bottom layer. Each well was fed once a week with 1 ml 0.33% noble agar in DFCI-1 medium. The wells were scored for colony growth over the next 4 weeks. Colonies constitute cell clumps greater than 60 μm in diameter.

Western blot analysis

Cells were lysed for 30 min on ice in a buffer containing 10 mM Tris- HCl, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.5% NP-40, 1 mM PMSF, 50 μg/ml aprotinin and 10 μg/ml leupeptin, pH 7.4. After removing the insoluble material, aliquots of supernatant containing 20 μg of protein were run through a 12% polyacrylamide gel under reducing conditions. Proteins were transferred to a immobilon-PSQ™ membrane (Millipore, Billerica, MA) that was subsequently incubated for overnight at 4°C in TBST buffer (10 mM Tris-HCl, 150 mM NaCl, 0.1% Triton X-100 and 0.05% Tween®20, pH 8.0) containing 5% skim milk. The membrane was incubated with c-H-ras monoclonal mouse IgG and actin monoclonal mouse IgG (Oncogene, Cambridge, MA). Binding of the antibodies was detected with the SuperSignal® West Pico Trial Kit (Pierce, Rockford, IL)

Statistical analysis

The values are presented as means ± S.E.M. A mixed model ANOVA was used to assess the overall level of significance across experimental groups. The within-subject variable was cross days and the between-subject variables were cell lines. Unpaired t-tests were performed to compare mean differences between 2 different groups. A p value of less than 0.05 was considered to be statistically significant. SSPS software was used for statistical analysis.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References

FISH confirms the rearrangement of chromosome 1to be 1q22-23

To determine the precise location of the rearrangement on chromosome 1 that underlies the phenotype of RAO-3 cell line, the BAC clone probes that correspond to human Chr.1q22-31 were used for FISH analysis in RAO-3 cell line. The probe-binding sites suggested that the chromosome rearrangement point was located on human chromosome 1q22-23 locus (Fig. 1). We propose that a segment of chromosome 1 from 1q22 to 1q23 has been duplicated and this duplicated segment has been inverted. The breakpoint is at 1 Chr.1q22.

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Figure 1. FISH of RAO-3 cell line. All probes were BAC clones from BPRC. (a) 8D14 BAC clone hybridizes to human chromosome 1q22. (b) 165A08 BAC clone hybridizes to 1q23-24. (c) 184N12 BAC clone hybridizes to1q24. (d) 196B07 hybridizes to1q32. The probes were labeled with the Bio-Nick Labeling System (Invitrogen, Gaithersburg). The results were observed under fluorescence microscopy with a FITC filter. Arrows show hybridization points on the chromosome 1. (e) The FISH study confirms that there is a duplication and an inversion from 1q22 to 1q23. The breakpoint is at 1q22.

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Loss of RAB25 expression in breast cancer cell lines

RAB25 expression was estimated by RT-PCR in series of breast cell lines. The band density of PCR products did not vary in the nontumorigenic cell lines RAO-1, RAO-2, HMEC6, HMEC6/RAS 61L, HMEC25 and HMEC26. The expression of RAB25 was also present in MX-1, MDA-MB-468, T-47D, MCF-7 and ZR-75-1 cancer cell lines; however, it was not detected in breast cancer cell lines RAO-3, RAO-4, MDA-MB-231 and MDA-MB-435s (Fig. 2a) (Table II). The housekeeping gene, β-actin, was used as a control for variation in loading.

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Figure 2. Loss of RAB25 gene mRNA expression in human breast cancer cell lines. RTPCR was performed on candidate genes. (a) Expression of RAB25 in human mammary cell lines. There is a consistent loss of RAB25 in the RAO-3, RAO-4 and MDA-MB-231 which all contain a RAS point mutation. (b) Expression of other RAB family members, RAB11a and RAB11b, remains unchanged. (c) Expression of RAB25 in tumorigenic (RAO-3, RAO-4) or non-tumorigenic (RAO-1, HMEC26) cell lines were examined using real-time PCR with fluorescence detection. The columns and the vertical bars represent the mean and the SEM, respectively. RAO-3 and RAO-4 expressed ∼1/6,000 of the level of RAB25 of RAO-1*. HMEC26 expressed comparable levels of RAB25 to RAO-1. *Statistical analysis found the data to be very significant (p < 0.01).

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Table II. Loss of RAB25 and RAS Mutation
Name of cell lineRAS mutationRAB25 expressionER/PR receptor
RAO-4H-RAS Q61L
RAO-3H-RAS Q61L
MDA-MB-231K-RAS G13D
MDA-MB-435s±
MX-1+
MDA-MB-468+
T-47D++
MCF-7++
ZR-75-1++

qPCR was used to confirm and quantify differences in RAB25 that were revealed by RT-PCR. The target transcripts were normalized to an endogenous housekeeping transcript, β-actin. The expression of RAB25 was drastically decreased in breast cancer cell lines RAO-3 and RAO-4 as compared to non-tumorigenic cell lines RAO-1 and HMEC26. RAB25 expression was reduced by ∼1:6,000 in the RAO-3 cell line as compared to RAO-1 (Fig. 2c). These results revealed that RAB25 expression was effectively lost in some breast cancer cell lines, but not in nontumorigenic cell lines.

Expression of 2 other genes RAB11a and RAB11b, having a strong homology with RAB25, was also evaluated. However, obvious differences in mRNA expression of these genes were not detected between nontumorigenic and tumorigenic cell lines (Fig. 2b).

RAB25 does not change the expression of RAS in cancer cell lines

To study whether forced expression of RAB25 in cancer cell lines would alter the expression of RAS, western blots were examined (Fig. 3). The expression of RAS was not changed after forced expression of RAB25 in the RAO-3 or MDA-MB-231 cell lines.

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Figure 3. Western blot analysis of RAS expression: c-H-ras monoclonal mouse IgG (lower) and actin monoclonal mouse IgG (upper) (Oncogene) were used as primary antibodies. Lane 1, RAO-1; lane 2, HMEC 6; lane 3, RAO-4; lane 4, RAO-3; lane 5, RAO-5/pLXSP; lane 6, RAO-3/RAB25; lane 7, MDA-MB-231; lane 8, MDA-MB-231/pLXSP and lane 9, MDA-MB-231/RAB25. There is no expression difference of RAS before or after transduction of RAB25.

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Transduction of RAB25 to cancer cell lines reversesin vitro transformation

To study the effects of RAB25, we transduced RAB25 into the RAO-3 cell line. Cell morphology changed in the transduced population, with a clear decline in focus formation (Fig. 4a). In the first 4 days, anchorage-independent growth and cell focus formation did not appear in RAO-3/RAB25 transformation cell line (Fig. 4a -a, e), whereas anchorage-independent growth and cell focus formation was obvious in RAO-3 (Fig. 4a-c, g) and RAO-3/pLXSP cell lines (Fig. 4a-b, f). The cell growth was significantly decreased in RAO-3/RAB25 cell line as compared to the parental RAO-3 line and RAO-3 with pLXSP alone (Fig. 4b). ANOVA analysis showed that viable cell number varied as a function of time and cell lines (ANOVA for within-subject variables: mean effect of time, F = 255.527, p < 0.001; time/cell lines interaction, F = 7.326, p < 0.001) (ANOVA for between-subject variables: mean effect of cell lines, F = 24.193, p = 0.001). On day 1, the cell number was not altered markedly between the 3 cell lines. However, from day 2, cell number was much lower in RAO-3/RAB25 cell line as compared to RAO-3 cell line (Fig. 4b). Otherwise, a significant difference was detected in cell number of RAO-3/RAB25 and RAO-3/pLXSP cell lines in days 2 and 3. Similar results were obtained in total cell number. Alteration of morphology and cell proliferation in RAO-3/RAB25 cell line is similar to the RAO-3 cell line treated with FTI-277 (Fig. 4a-d, h and 4c).

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Figure 4. (a) Morphology of cells. The pictures were taken 96 hr after passage. a, e: RAO-3/RAB25 cell line. Cell focus formation and anchorage-independent growth were lost after transduction of RAB25 to the RAO-3 tumorigenic cell line. b, f: RAO-3/pLXSP cell line. c, g: RAO-3 cell line. d, h: RAO-3 cell line treated with 10 μM FTI-277 for 96 hr. Magnification: top ×100 and below ×400. (b), (c) Cell count: the columns and the vertical bars represent the mean and the SEM, respectively. The forced expression of RAB25 caused significant reduction in the cell growth with reduced viable cell numbers after 2 days of cell culturing. The cell number of RAO-3/RAB25 greatly lags behind RAO-3 and RAO-3/pLXSP (b). Similar results were obtained when the RAO-3 cell line was treated with 10 μM FTI-277 (c). *Shows a significant statistical difference (p < 0.01) between RAO-3 and RAO-3/RAB25 cell lines (b) or between RAO-3 control and RAO-3 after FTI-277 treatment (c).

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The soft agar assay was used to observe the cell anchorage-independent growth and ability of tumorigenesis. After incubation at 37°C for 4 weeks, the growth state of cell multiple colony formation showed a marked difference. A number of clusters developed on the soft agar plates with RAO-3 and RAO-3/pLXSP cells (Fig. 5a-a, d), but not on the soft agar plates with RAO-1 and RAO-3/RAB25 transformation cells (Fig. 5a-b, c). This situation remained for 10 weeks until the plates were destroyed. These results suggested that RAB25 could reverse the transformation by the Q61L mutant RAS.

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Figure 5. Soft agar assay. (a) The RAO-3 cell line was used as a positive control (a), and the RAO-1 cell line was used as a negative control (b). No cell colony formation was seen on the plate of the RAO-3/RAB25 cell line (c), but colonies developed on the plate of the RAO-3/pLXSP cell line (d). The picture was taken at 4 weeks. (b) Cell colony count: the number of cell colonies was remarkably different between RAO-3 and RAO-3/RAB25 cell line.

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Expression of RAB25 in breast tissue and loss of RAB25 in some breast cancer tissues

RAB25 expression was examined in human breast tissue. The primary mammary epithelial cells were divided from mammary tissues that were collected from mammoplasty samples of 2 healthy donors. Expression of RAB25 was also tested in uninvolved breast tissues from breast cancer patients and breast cancer tissues by RT-PCR. The data show that RAB25 expression was present in normal human breast tissue and human primary mammary epithelial cells growth in monolayer (Fig. 6a and 6b), but not in some breast cancer tissues (Fig. 6b). A total of 18 breast cancer tissues from patients were tested. Loss of RAB25 was detected in 6 cancer tissues. This result suggests that expression of RAB25 is lost in about 33% of breast cancer tissues.

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Figure 6. Expression of RAB25 in human mammary tissue: (a) expression of RAB25 in breast tissue. HMEC JC4 and HMEC JC5 are primary HMEC coming from healthy donors undergoing reduction mammoplasties. RAB25 is expressed in primary HMEC. (b) Loss of expression of RAB25 in breast cancer tissue. Lane 1, 1K DNA ladder; lanes 2, 4, 6: normal, matched breast tissues; lanes 3, 5, 7: tumor tissue from 3 patients. RT-PCR shows that expression of RAB25 is retained in matched normal tissue while it is lost in tumor tissue.

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Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References

RAS (H-RAS, K-RAS, N-RAS) is a signal-transducing small GTPase that plays a central role in the control of cell growth and differentiation.19, 20 Mutations in RAS genes have all been found in human tumors, and the frequency of RAS mutations is the highest among any genes in human cancers.21, 22 The mutation at the Q61L position of RAS is one of the most common in naturally occurring neoplasia and experimentally induced animal tumors.9, 10 The RAS-transformed fibroblasts display typical anchorage-independent growth and morphological changes. These phenotypes are thought to be caused by the RAS-induced gene expression and the rearrangement of the cytoskeleton and the cell adhesion proteins.23 We created a novel series of breast cancer cell lines, RAO-3 and RAO-4, by transduction of the Q61L mutant H-RAS to RAO-1, which is an immortalized HMEC line established by hTERT transduction.4 Expression of RAB25, which is located on chromosome locus 1q22, was lost in some of the breast cancer cell lines and the breast cancer tissue from patients, but not in the nontumorigenic cell lines and the normal tissues (Figs. 2a and 6). In particular, RAB25 expression was maintained in the RAO-1 and the RAO-2 cell lines. RAO-2, although nontumorigenic, is derived from RAO-1 and overexpresses the Q61L mutant H-RAS. This suggests the loss of RAB25 is critical to the transformation process and is not the result of H-RAS suppression. After transduction of RAB25 to the RAO-3 cell line, RAS levels remained unaltered (Fig. 3). However, cell proliferation was inhibited, cell focus formation was reduced (Fig. 4), and the ability to form colonies in soft agar was remarkably decreased in RAO-3/RAB25 cell line as compared to RAO-3 cell line (Fig. 5).

The RAB proteins cycle between an active, GTP-bound form, and an inactive, GDP-bound form. The GTP-bound form is able to interact with effector molecules and transmit downstream signals.24, 25, 26, 27, 28 Gene knock-out studies in yeast have shown that some RAB GTPases are essential, whereas others are dispensable,29 and some genetic diseases have been associated with RAB GTPases or their interacting proteins.30

A number of studies have demonstrated the over expression of RAS at the mRNA and protein level in breast cancer, although the incidence of RAS point mutations in primary breast cancer is rare (<5%).31, 32, 33 The Q61L mutation of RAS results in an oncogenic variant that is insensitive to GAP and persists in the GTP-bound state and exhibits dominant transformation activity.34

The direct relationship between RAS-GTP and transformation has been demonstrated.34 Loss of expression of RAB25 in the RAO-3 cell line, but not in the nontumorigenic RAO-2 cell line, suggests that a relative deficiency of RAB25 may be favorable for the transformation activity of mutant RAS in the RAO-3 cell line. Furthermore, transduction of RAB25 caused the inhibition of cell focus formation in the RAO-3 cell line, implying that RAB25 impairs or blocks the RAS signaling pathway. This result is very similar to the results seen when the RAO-3 cell line was treated with FTI-277 (Fig. 4a-d, h and 4c), a farnesyltransferase (FTase) inhibitor. In RAS activation, the first and most critical modification is the addition of a farnesyl isoprenoid moiety in a reaction catalyzed by the enzyme protein FTase. It follows that inhibiting FTase would prevent RAS from maturing into its biologically active form.35

Interestingly, the GTP-binding site of RAB25, DTAGLE, is identical to the GTP-binding site of Q61L mutant RAS in the switch II region, which is the active center of the molecule and is involved in the binding interaction between RAS/RAB25 and GTP.12, 27, 36 This identity might produce a competition for locally available GTP between RAB25 and mutant RAS. We hypothesize that transduction of RAB25 prevented RAS from reaching an activated form and decreased the RAS GTP-bound form, which, in turn, reversed morphological changes and inhibited cell proliferation. Loss of RAB25 may be necessary for RAS-driven breast cancer tumorigenesis, particularly when the Q61L H-RAS mutant is involved.

RAS activity can lead to proapoptotic or antiapoptotic responses.37 The complex nature of the transformation phenotype caused by oncogenic RAS might require RAS activation of multiple signaling pathways. There are 3 major downstream factors, Raf serine/threonine kinases, phosphoinositide 3-kinases (PI3Ks)/AKT (protein kinase B) and RalGDS, in RAS signaling pathways.38, 39, 40 Different mutations of RAS produce distinct impacts on these signaling pathways, and certain RAS-driven tumors may require the loss of RAB25 to promote tumorigenesis to fully activate all of the downstream pathways.37 Recently, increased RAB25 expression was reported in breast and ovarian cancer by Cheng et al., and an interaction between RAB25 and PI3K pathway was described.41 It suggests a pathological role for RAB25 in epithelial tumor development. The study by Cheng et al.41 found increased expression of RAB25 in 47% and 66.7% of breast tumor samples that were analyzed by array CGH and microarray, respectively. However, the methodology does not permit the detection of mutations in the RAB25 genome which may lead to inactivation or a dominant negative phenotype. Additionally, the functions of RAB25 overexpression were tested on only one breast cancer cell line, MCF-7, which is estrogen and progesterone receptor positive. In this study, we tested fifteen cell lines of which 6 were nontumorigenic and 9 were tumorigenic. RAB25 was detected in all non-tumorigenic cell lines, and loss of RAB25 was noted in 4 tumorigenic cell lines (Fig. 2) (Table II). Loss of RAB25 expression was also detected in about 33% human breast cancer tissues. Just like RAS, RAB25 may cause a diverse spectrum of cellular responses that might require multiple effectors. RAB25 may be similar to p53, which was first described as an oncogene only,42, 43, 44 later to be rediscovered as a tumor suppressor.

We report the first link between RAB25 and RAS in breast cancer. Loss of RAB25 was exclusively seen in estrogen receptor (ER) and progesterone (PR)-negative tumorigenic mammary cell lines and strongly correlated with mutations in the RAS oncogene. In our clinical tumor samples, only 8% (1/12) of tumors that were ER or PR positive lost RAB25 whereas 83% (5/6) of the ER and PR negative tumors displayed loss of RAB25. Our in vitro data suggests that RAB25 loss is occurring in a subset of hormonally insensitive mammary tumors that is harboring a RAS mutation. The possibility that RAB25 has dual actions in breast cancer has emerged. We hypothesize that RAB25 expression is maintained in a “homeostatic” range. Perturbation of these levels in the appropriate molecular context may lead to mammary cell transformation. Our data provide new insight in understanding the cofactors of RAS that mediate mammary cell transformation at the molecular level and may help to develop novel treatment strategies.

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References