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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.
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- Material and methods
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.