Rac-GAP-dependent Inhibition of Breast Cancer Cell Proliferation by β2-Chimerin*

β2-Chimerin is a member of the “non-protein kinase C” intracellular receptors for the second messenger diacylglycerol and the phorbol esters that is yet poorly characterized, particularly in the context of signaling pathways involved in proliferation and cancer progression. β2-Chimerin possesses a C-terminal Rac-GAP (GTPase-activating protein) domain that accelerates the hydrolysis of GTP from the Rac GTPase, leading to its inactivation. We found that β2-chimerin messenger levels are significantly down-regulated in human breast cancer cell lines as well as in breast tumors. Adenoviral delivery of β2-chimerin into MCF-7 breast cancer cells leads to inhibition of proliferation and G1 cell cycle arrest. Mechanistic studies show that the effect involves the reduction in Rac-GTP levels, cyclin D1 expression, and retinoblastoma dephosphorylation. Studies using the mutated forms of β2-chimerin revealed that these effects were entirely dependent on its C-terminal GAP domain and Rac-GAP activity. Moreover, MCF-7 cells stably expressing active Rac (V12Rac1) but not RhoA (V14RhoA) were insensitive to β2-chimerin-induced inhibition of proliferation and cell cycle progression. The modulation of G1/S progression by β2-chimerin not only implies an essential role for Rac in breast cancer cell proliferation but also raises the intriguing possibility that diacylglycerol-regulated non-protein kinase C pathways can negatively impact proliferation mechanisms controlled by Rho GTPases.

␤2-Chimerin is a member of the "non-protein kinase C" intracellular receptors for the second messenger diacylglycerol and the phorbol esters that is yet poorly characterized, particularly in the context of signaling pathways involved in proliferation and cancer progression. ␤2-Chimerin possesses a C-terminal Rac-GAP (GTPase-activating protein) domain that accelerates the hydrolysis of GTP from the Rac GTPase, leading to its inactivation. We found that ␤2-chimerin messenger levels are significantly down-regulated in human breast cancer cell lines as well as in breast tumors. Adenoviral delivery of ␤2-chimerin into MCF-7 breast cancer cells leads to inhibition of proliferation and G 1 cell cycle arrest. Mechanistic studies show that the effect involves the reduction in Rac-GTP levels, cyclin D1 expression, and retinoblastoma dephosphorylation. Studies using the mutated forms of ␤2-chimerin revealed that these effects were entirely dependent on its C-terminal GAP domain and Rac-GAP activity. Moreover, MCF-7 cells stably expressing active Rac (V12Rac1) but not RhoA (V14RhoA) were insensitive to ␤2-chimerin-induced inhibition of proliferation and cell cycle progression. The modulation of G 1 /S progression by ␤2-chimerin not only implies an essential role for Rac in breast cancer cell proliferation but also raises the intriguing possibility that diacylglycerol-regulated non-protein kinase C pathways can negatively impact proliferation mechanisms controlled by Rho GTPases.
Chimerins represent a family of four closely related GAPs 1 (GTPase-activating proteins) for small GTPases that were orig-inally characterized as high affinity intracellular receptors for the second messenger diacylglycerol (DAG) and the phorbol ester tumor promoters (1)(2)(3)(4). Structurally, chimerins possess a C1 domain highly homologous to those of PKC isozymes (the DAG/phorbol ester binding site) and a C-terminal GAP domain. The ␣2and ␤2-chimerins also have a N-terminal Src homology 2 domain of unknown function, which is not present in the splice variants ␣1-(or n-) and ␤1-chimerins (5,6). Very little information is available regarding the regulation, expression, and function of ␤2-chimerin or the other chimerin isoforms as well as their role in proliferation mechanisms and cancer progression. We have been focusing our attention on ␤2-chimerin, because there is emerging evidence that this isoform is directly regulated by phorbol esters (4,7) as well as tyrosine-kinase receptors that couple to DAG generation. 2 Importantly, early studies in gliomas have suggested a potential role for ␤2-chimerin as a tumor suppressor (8) but its relevance in other cancer models is still unknown.
In vitro studies have shown that the C-terminal domain of chimerins is capable of accelerating GTP hydrolysis from the small GTPase Rac1 without affecting the activity of RhoA or Cdc42 GTPases (9,10). Our recent studies in COS cells revealed that ␤2-chimerin decreases cellular Rac-GTP levels and inhibits the elevation of Rac-GTP levels caused by epidermal growth factor (EGF) (10,11). Rac GTPase is known to act as a molecular switch, cycling between an active GTP-bound state (Rac-GTP) and an inactive GDP-bound state (Rac-GDP). This switch is regulated by three groups of molecules: 1) guanine nucleotide exchange factors, such as Vav and Tiam-1, that promote its conversion to the active GTP-bound form; 2) guanine nucleotide dissociation inhibitors; and 3) GAPs, which stimulate intrinsic GTPase activity, thus leading to Rac inactivation (12,13). Active Rac interacts with various effectors to initiate downstream signaling events that control the dynamics of actin cytoskeleton reorganization, migration, adhesion, and gene expression (14 -17). Rac and Rac-guanine nucleotide exchange factors play key roles in the control of various aspects of malignant transformation and the metastatic cascade in various models, including breast cancer cells (18 -20). Several laboratories (21)(22)(23)(24)(25) have proposed a role for Rac in the control of mitogenesis through its ability to regulate G 1 /S transition and cyclin D1 expression. Moreover, Rac and other members of the Rho GTPase family are overexpressed in human tumors such as in breast cancer (26,27) and hyperactivation of Rac leading to a higher rate of cell proliferation has been found in cellular models of human breast cancer (28). Targeted expression of an activated Rac mutant in mammary epithelium causes mam-* This work was supported by Grants RO1-CA74197 (National Institutes of Health) and RPG-97-092-06-CNE (American Cancer Society) (to M. G. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  mary gland lesions (29). In addition, there is strong evidence that Rac effectors such as p21-activated kinase 1 are dysregulated in breast cancer cells (30). Collectively, these findings suggest critical implications of Rac in tumorigenesis, particularly in models of breast cancer.
In this paper, we investigated the expression of the DAG/ phorbol ester receptor ␤2-chimerin in breast cancer and its role in proliferation. We have found that ␤2-chimerin mRNA levels are strikingly reduced in breast cancer cell lines and tissues. By means of adenoviral delivery into MCF-7 breast cancer cells, we have found that ␤2-chimerin, but not the mutated forms lacking Rac-GAP activity, causes a significant impairment in G 1 /S cell cycle progression due to a reduction in the expression levels of cyclin D1. The effect of ␤2-chimerin is strictly dependent on its ability to inhibit Rac function via the C-terminal GAP domain, suggesting the possibility that Racmediated control of cell proliferation is modulated by DAGregulated pathways.

EXPERIMENTAL PROCEDURES
Human Breast Non-malignant and Cancer Cell Lines-Human breast cancer cell lines MCF-7, T-47D, MDA-MB231, MDA-MB-435, MDA-MB-468, Hs578T, and human breast immortalized non-malignant MCF-10A cells were purchased from ATCC and cultured as recommended by the provider. The human non-malignant breast cell line HMT-3522 and its malignant derivative T4-2 were cultured as previously described (31). MCF-7-Tet-On cells were purchased from Clontech and cultured in DMEM supplemented with 10% FBS, 2 mM glutamine, and 100 g/ml G418.
Examination of ␤2-Chimerin mRNA Levels in Human Breast Cells and Tissues-For tissue RNA, 10 pairs of high quality human breast cancer tissue total RNA and matched-normal tissue total RNA (from the same patient) were purchased from Clinomix Biosciences, Inc. (Watervliet, NY). All 10 patients were diagnosed as infiltrating ductal carcinoma (IDC) at different stages including two Stage I IDCs (samples 1-2), two Stage II IDCs (samples 3-4), two Stage III IDCs (samples 5-6), and four Stage IV IDCs (samples 7-10). Total RNA was prepared using TRIzol and reversibly transcripted using SuperScript TM II Reverse Transcriptase (Invitrogen). ␤2-Chimerin mRNA levels were determined either by standard PCR (30 cycles) using the following primers: 5Ј-TGATCTCAAGAGGATCAAGAA-3Ј (forward) and 5Ј-TTG-GAATAGGTATCATATGTG-3Ј (reverse), which specifically amplify a 297-bp fragment of ␤2-chimerin. Primers used for real-time PCR (Q-PCR) are described elsewhere (32). The real-time PCR reactions were plated in triplicate and performed in 384-well plates using the ABI 7900HT sequence detection system (Applied Biosystems, Foster City, CA). Glyceraldehyde-3-phosphate dehydrogenase was used for normalization (32).
Generation of Adenoviruses (AdVs)-AdVs were generated with the AdEasy TM adenoviral vector system (Stratagene). Generation of the ␤2-chimerin and ␤-GAP AdVs was described elsewhere (10,32). For the generation of ⌬EIE-␤2-chimerin adenoviral construct, a XhoI-MluI insert comprising the mutant ⌬EIE-␤2-chimerin (10) was ligated into pShuttle-CMV-HA. A similar strategy was used for the generation of an AdV for the mutant I130A-␤2-chimerin (33). A control LacZ-AdV was generated from pShuttle-CMV-LacZ (provided by the kit) and therefore has the same backbone as the ␤2-chimerin AdVs. For adenoviral infections, MCF-7 cells in 6-well plates growing in serum-free DMEM were infected with various AdVs for 16 h. AdVs were removed after extensive washing, and experiments were performed 48 h later.
Cell Proliferation and Cell Cycle Analysis-Cell proliferation was assessed by BrdUrd incorporation and by the MTS assay (CellTiter 96® Aqueous One solution cell proliferation assay, Promega). After overnight infection (16 h) with the different AdVs, cells were washed once with PBS and incubated in serum-free DMEM for 24 h and then cultured in DMEM supplemented with 10% FBS for another 24 h. BrdUrd (Sigma) was then added into the medium for 30 min (final concentration: 0.2 mM). Cells were then collected by trypsinization, washed with PBS, and fixed with 70% ethanol for BrdUrd incorporation analysis using flow cytometry (35). For the MTS assay, after overnight adenoviral infection in 10-cm dishes, cells were collected using trypsin, counted, and then seeded onto 96-well plates (1 ϫ 10 4 cells/well in 100 l of DMEM supplemented with 10% FBS). MTS was added after 24, 48, or 72 h, and absorbance was measured at 490 nm (36). For cell cycle analysis, after overnight (16 h) adenoviral infection, cells were washed once with PBS, incubated in serum-free DMEM for 24 h, and then cultured in DMEM supplemented with 10% FBS for another 24 h. Cells were then collected by trypsinization and analyzed using flow cytometry as previously described (37).
Rac-GTP and Cdc42-GTP Pull-down Assays-After overnight infection (16 h) with different AdVs, cells were incubated in serum-free DMEM for 24 h and then stimulated with EGF (100 ng/ml, 1 min). Alternatively, after adenoviral infection, cells were cultured in 10% FBS DMEM for an additional 24-h period. Rac-GTP and Cdc42-GTP levels were determined with a "pull-down" assay using the PBD (p21-binding domain) of p21-activated kinase, as previously described (10,11), and using either anti-Rac or anti-Cdc42 antibodies for detection, respectively.
Statistical Analysis-Data were analyzed using either a Student's t test or one-way analysis of variance (ANOVA) with Scheffe's test. A p value of Ͻ0.05 was considered statistically significant.

Reduced Expression of ␤2-Chimerin in Human Breast Cancer
Cells and Tissues-The expression of ␤2-chimerin in normal and breast cancer cells is unknown. Using standard PCR analysis, we found high levels of ␤2-chimerin mRNA in non-malignant immortalized MCF-10A cells. On the other hand, in all of the cancer cell lines examined, the ␤2-chimerin transcript was barely detected or dramatically reduced (Fig. 1A). The results were confirmed by a quantitative analysis using Q-PCR. Indeed, ␤2-chimerin mRNA was not detected in MCF-7 and Hs578T cells and it was very low in T-47D, MDA-MB-231, and MDA-MB-435 cells. Only MDA-MB-468 cells showed significant levels of ␤2-chimerin transcript, although much lower than MCF-10A cells (Fig. 1B). Similarly, whereas ␤2chimerin mRNA was readily detected in the non-malignant breast cell line HMT3522 (31), it was barely detectable in its malignant derivative (T4-2) (Fig. 1, A and B). We next examined ␤2-chimerin mRNA levels in a small sample of human breast cancer tissues and their corresponding matched-normal tissues (from the same patient). It was found that the expression of ␤2-chimerin mRNA in normal tissues was highly variable. However, among the 10 patients, the ␤2-chimerin transcript was significantly lower in the cancer tissues of 7 patients (Fig. 1C). Together, these results reveal a significant reduction of ␤2-chimerin expression in human breast cancer.
␤2-Chimerin Reduces Cyclin D1 Expression and Inhibits pRb Phosphorylation-Because expression of ␤2-chimerin in MCF-7 cells leads to G 1 /S arrest, we next assessed the effect of ␤2-chimerin on pRb phosphorylation. The expression of ␤2chimerin dose-dependently reduced pRb phosphorylation (Fig.  4A). Notably, ␤2-chimerin significantly inhibited the expression of cyclin D1. ␤2-Chimerin also reduced the expression of cyclin A and caused a slight increase in cyclin E levels. On the other hand, cells infected with control LacZ-AdV (100 m.o.i.) showed no obvious alterations in cyclin expression and pRb phosphorylation. Infection of MCF-7 cells with ␤2-chimerin-AdV did not cause any significant changes on the expression of cyclin-dependent kinases 2, 4, and 6 (data not shown). Infection of MCF-7 cells with the ␤-GAP-AdV also led to a reduction in cyclin D1 levels and pRb phosphorylation (Fig. 4A). However,

␤2-Chimerin Inhibits Breast Cancer Cell Proliferation
the Rac-GAP inactive mutant, ⌬EIE-␤2-chimerin, did not impair cyclin D1 expression or pRb phosphorylation (Fig. 4B). Taken together, these results suggest that the inhibition of G 1 /S transition by ␤2-chimerin via its ␤-GAP domain involves the reduction of cyclin D1 and pRb phosphorylation levels.
Inhibition of Rac by ␤2-Chimerin in MCF-7 Cells-␤2-Chimerin has specificity for the Rac GTPase both in in vitro GAP assays and in COS-1 cells but does not affect RhoA or Cdc42 activity (10,11). EGF (100 ng/ml, 1 min) caused a 3.3 Ϯ 0.5-fold (n ϭ 3) increase in Rac-GTP levels in MCF-7 cells, which was significantly impaired by the expression of ␤2 chimerin. ␤2-Chimerin also reduced Rac-GTP levels in MCF-7 cells growing in 10% serum. The effect was proportional to the m.o.i. used for infection, and it was not observed with the GAP-inactive mutant, ⌬EIE-␤2-chimerin (Fig. 5A). A densitometric analysis of the ␤2-chimerin effect on serum-induced activation of Rac is presented in Fig. 5B. A striking correlation was observed between the inhibitory effect of ␤2-chimerin on Rac activity and the reduction in cyclin D1 levels by different m.o.i. of the ␤2-chimerin-AdV (r ϭ 0.95).
Ectopic Expression of Cyclin D1 Rescues the Anti-proliferative Effect of ␤2-Chimerin-To further explore the link between Rac and cyclin D1 in our experimental model, we expressed cyclin D1 using a retroviral approach (Fig. 6A). Interestingly, the ectopic expression of cyclin D1 using the D1-RetroV significantly rescued the anti-proliferative effect of ␤2-chimerin, whereas the control retrovirus (V-RetroV) did not (Fig. 6B).
We then determined whether the expression of other active Rho-GTPases could rescue the effect of ␤2-chimerin. MCF-7 cell lines stably expressing constitutively active Cdc42 (V12Cdc42) or RhoA (V14RhoA) were generated (Fig. 7A). Similar to control MCF-7 cells, HA-V14RhoA-MCF-7 cells were highly sensitive to ␤2-chimerin for the inhibition of cell proliferation, reduction of cyclin D1, and Rb dephosphorylation (Fig.  7, A and B). Unexpectedly, in cells expressing active Cdc42,

␤2-Chimerin Inhibits Breast Cancer Cell Proliferation
adenoviral delivery of ␤2-chimerin was unable to inhibit cell proliferation, cyclin D1 expression, and pRb phosphorylation (Fig. 7, A and B). To further examine the mechanisms involved in the protective effect of V12Cdc42, we determined Cdc42-GTP levels in response to EGF (100 g/ml, 1 min). A 3.1 Ϯ 0.6-fold (n ϭ 3) increase in Cdc42-GTP levels was observed in response to the growth factor, which was not affected by the ␤2-chimerin-AdV, even at the highest m.o.i. used (100 plaque-forming units/cell) (Fig. 7C). Interestingly, we found that basal Rac-GTP levels were elevated in HA-V12Cdc42-MCF-7 cells (Fig. 7,  D and E), which probably explain the protective effect of active Cdc42 on ␤2-chimerin-induced inhibition of cyclin D1 levels, pRb phosphorylation, and cell proliferation.
A Hyperactive ␤2-Chimerin Mutant Is a Potent Inhibitor of Cyclin D1 Expression and Proliferation-Based on structural predictions gained from the recently solved structure of ␤2chimerin, we generated an AdV encoding for a ␤2-chimerin mutant locked in the constitutively active conformation. This mutant, I130A-␤2-chimerin, was shown to have constitutive Rac-GAP activity when expressed in COS-1 cells by bypassing lipid activation (33). An AdV encoding for I130A-␤2-chimerin was generated and used to infect MCF-7 cells. We optimized conditions to achieve similar low levels of expression as those observed in the non-malignant breast cell line HMT3522 cells (as detected by Q-PCR, Table I). In this case, we used a lower m.o.i. and shorter expression times (16 h instead of 40 h) and the levels of the mutant I130A-␤2-chimerin in MCF-7 cells were well below the detection levels using Western blot. Under these experimental conditions, the wild-type ␤2-chimerin still showed very high levels of expression by Western blot and caused a ϳ25% reduction in Rac-GTP levels (Fig. 8A). Remark-

␤2-Chimerin Inhibits Breast Cancer Cell Proliferation
ably, even if I130A-␤2-chimerin was expressed at very low levels, it caused a 51% reduction in Rac-GTP levels (Fig. 8A). Moreover, I130A-␤2-chimerin markedly reduced cyclin D1 levels (Fig. 8B) and impaired cell proliferation (Fig. 8C). DISCUSSION Understanding the functional properties of Rac-GAPs is relevant, because Rac is a key player in the process of malignant transformation and metastasis (12,18,30). The two most relevant findings in the present study are that ␤2-chimerin expression is down-regulated in breast cancer and that the expression of this Rac-GAP in MCF-7 breast cancer cells impairs G 1 /S cell cycle progression by reducing cyclin D1 levels and Rb phosphorylation. Inhibition of proliferation by ␤2-chimerin in MCF-7 cells is dependent on the ␤2-chimerin GAP activity, and indeed, a functionally active GAP domain is required for the anti-mitogenic effect. Our results suggest that Rac activity is critical for G 1 /S progression in breast cancer MCF-7 cells.
Analysis of ␤2-chimerin mRNA levels revealed that breast cancer cells have significantly lower levels than non-malignant cells. This effect is particularly striking when we compare the non-malignant HMT3522 cell line with its malignant derivate T4-2 cell line. Moreover, studies using matched pairs of RNA samples from breast cancer patients revealed that ␤2-chimerin is significantly down-regulated in 70% of tumor samples. Members of the Rho GTPase family such as Rac and Rho are overexpressed in human tumors, particularly in breast cancer (26,27). Rac activity was found to be elevated in transformed cells, as recently described in v-Src-transformed fibroblasts, and inhibition of Rac function using dominant-negative Rac mutants dramatically reduced the ability of v-src to transform NIH 3T3 cells (39). Small GTPase hyperactivation may be the consequence of enhanced upstream inputs and/or reduced activity of GAPs, as suggested by Mira and co-workers (28) in breast cancer cell models. Various mechanisms can account for the elevated upstream inputs including receptor hyperactivation and/or enhanced activation of Rac-guanine nucleotide exchange factors, such as Tiam1 and Vav (20,40). On the other hand, the relative contribution of the down-regulation of Rho GAPs in cancer progression and their potential roles as tumor suppressors has not been extensively studied. For example, a recent study (41) has found that the Rho/Cdc42 GAP DLC2 is

␤2-Chimerin Inhibits Breast Cancer Cell Proliferation
significantly underexpressed in 18% human hepatocellular carcinoma. It is conceivable that the down-regulation of ␤2-chimerin in breast cancer cells may contribute, at least in part, to the progression of the disease. Early studies in glioma models have identified ␤2-chimerin as a gene that is significantly down-regulated in high-grade gliomas compared with normal brain and low-grade astrocytomas (8). Down-regulation of ␤2chimerin in advanced stages of the disease could contribute to the enhanced proliferation and metastatic dissemination of glioma cells due to dysregulation of Rac activity. Along the same lines, we have recently found using tissue microarrays that ␤2-chimerin expression is reduced by ϳ60% in benign duodenal adenomas and ϳ80% in duodenal adenocarcinomas when compared with normal tissues. 3 ␤-GAP significantly inhibits cell migration as well as tumor growth, invasiveness, and metastatic dissemination in vivo (32), suggesting that specific inhibition of Rac by ␤2-chimerin may impinge on various steps of malignant transformation. Although more extensive studies would be required to establish whether this Rac-GAP may serve as a prognostic marker, this body of evidence suggests that down-regulation of ␤2-chimerin expression may contribute to breast cancer progression. This may also be relevant in tissues that express high levels of ␤2-chimerin, including brain, pancreas, and intestine. Adenoviral delivery of ␤2-chimerin, ␤-GAP, or I130A-␤2chimerin, but not ⌬EIE-␤2-chimerin, significantly impairs proliferation and elevations in Rac-GTP levels in MCF-7 breast cancer cells, suggesting an essential role for chimerin Rac-GAP activity in these effects. ␤2-Chimerin also impairs heregulin ␤1-induced Rac activation and proliferation in breast cancer cells. 4 This highlights the potential relevance of ␤2-chimerin as a general negative regulator of growth factor-mediated mitogenic responses. Moreover, we have recently observed that ␤2-chimerin RNAi in HeLa cells leads to a significant potentiation of EGF-induced Rac activation. 2 Our results also emphasize the importance of Rac in cell cycle control, as previously reported using constitutively active and dominant-negative Rac1 mutants (42)(43)(44). A dominantnegative N17Rac1 mutant impairs serum-induced DNA synthesis in fibroblasts and has been reported to causes cell growth arrest in G 1 (21) or G 2 /M (45). Although the specificity of dominant-negative Rac mutants may be questioned, our experiments revealed that the inhibition of Rac activity with a specific Rac-GAP leads to G 1 /S arrest in MCF-7 breast cancer cells, an effect that strongly correlates with the reduction in cyclin D1 and pRb phosphorylation. Rac regulates cyclin D1 expression, probably at multiple levels, depending on the experimental condition and cell type. For example, dominant-negative and constitutively active forms of Rac1 regulate cyclin D1 promoter activity in smooth muscle cells through Rac-dependent generation of reactive oxygen species (24). A role for the NF-B pathway downstream of Rac has also been described in NIH 3T3 cells (23). Regulation of the cyclin D1 messenger by Rac at a translational level has also been reported (21,25). Activated Rac is capable of enhancing pRb phosphorylation and E2F-mediated transcription of genes required for S phase entry and DNA replication (46). It has also been proposed that Rac integrates signals from specific integrins and growth factors to promote the synthesis of cyclin D1 and tumor cell survival (24,25,47). The insensitivity of V12Rac1-expressing MCF-7 cells to ␤2-chimerininduced reduction in cyclin D1 levels and Rb phosphorylation, as well as the rescue of the ␤2-chimerin effect by ectopic expression of cyclin D1, further supports the Rac-cyclin D1-G 1 /S progression link.
An emerging paradigm is that ␤2-chimerin can be regulated by cell surface receptors, as it is well known for Ras-GAPs (48). Receptors coupled to the generation of the lipid second messenger DAG, such as the EGF receptor, control the activity of ␤2-chimerin both by positional and allosteric mechanisms, 2 which substantiates the concept of DAG divergence via the activation of "non-PKC" pathways. These DAG-regulated mechanisms, as well as the selectivity of ␤2-chimerin for the Rac GTPase, have been further validated by the recently solved three-dimensional structure of this Rac-GAP (33). Our hypothesis is that, in the context of receptors such as the EGF or platelet-derived growth factor receptor, ␤2-chimerins represent a DAG-regulated negative loop that self-limits Rac activation and Rac-mediated responses. Our focus now is to elucidate the molecular basis of such lipid regulation, which will provide further insight into the receptor-mediated control of chimerin function and G 1 /S cell cycle progression in breast cancer and other diseases.