The aim of the present study was to explore the expression profile and the potential regulatory mechanism of two long non-coding RNAs (lncRNAs) (RP13-650J16.1 and TCONS_00023979) in prostate cancer (PCa). Expression profile of lncRNAs in PCa and paracancerous tissues were investgated by the high-throughput gene chip technology. Specific siRNA of RP13-650J16.1 or TCONS_00023979 was transfected into DU145 cells. Then, the relative expression of RP13-650J16.1, receptor-associated coactivator 3 (RAC3), promyelocytic leukemia (PML), and TCONS_00023979 was detected by quantitative real-time PCR and Western blotting. MTT assay was used to detect the proliferation of DU145 cells. The migration ability of DU145 cells was measured by Transwell chambers. Single cell proliferation and clonogenic ability were detected by plate clone formation assay. RP13-650J16.1 and RAC3 expression was up-regulated, and TCONS_00023979 and PML expression was down-regulated in PCa tissues. Silencing RP13-650J16.1 could decrease RAC3 expression, and knockout of TCONS_00023979 also reduced PML expression. Moreover, the ability of proliferation, migration, and colony formation of DU145 cells was decreased after transfected with si-RP13-650J16.1, while these abilities were increased after transfected with si-TCONS_00023979. Collectively, our findings demonstrated that RP13-650J16.1 might be an oncogene and TCONS_00023979 might be an antioncogene in PCa.

The initial step of metastasis is the local invasion of tumor cells into the surrounding tissue. Invadopodia are actin-based protrusions that mediate the matrix degradation necessary for invasion and metastasis of tumor cells. We demonstrate that Rac3 GTPase is critical for integrating the adhesion of invadopodia to the extracellular matrix (ECM) with their ability to degrade the ECM in breast tumor cells. We identify two pathways at invadopodia important for integrin activation and delivery of matrix metalloproteinases: through the upstream recruiter CIB1 as well as the downstream effector GIT1. Rac3 activity, at and surrounding invadopodia, is controlled by Vav2 and βPIX. These guanine nucleotide exchange factors regulate the spatiotemporal dynamics of Rac3 activity, impacting GIT1 localization. Moreover, the GTPase-activating function of GIT1 toward the vesicular trafficking regulator Arf6 GTPase is required for matrix degradation. Importantly, Rac3 regulates the ability of tumor cells to metastasize in vivo. The Rac3-dependent mechanisms we show in this study are critical for balancing proteolytic activity and adhesive activity to achieve a maximally invasive phenotype.

PURPOSE: Rac3 plays an important role in regulating tumorigenesis. Autophagy plays a vital role in tumorigenesis and tumor progression. The relationship between the two remains unclear. The objective of the present study was to determine the specific molecular mechanism of intracellular Rac3 in regulating autophagy and reveal the relationship between tumor cell autophagy and apoptosis.
METHODS: A laser confocal microscope was used to photograph the accumulated EGFP-MAP1LC3 spots for investigating the relationship between Rac3 and autophagy at the cellular level. Immunoblotting was also used to investigate the relationship between Rac3 and autophagy. The autophagy flux arising from inhibition of Rac3 was detected with autophagy inhibitors and ATG5 and ATG7 siRNA interference experiments. ATF4 and DDIT4 siRNA interference and overexpression experiments were conducted to investigate the relationship between endoplasmic reticulum stress, the MTOR signaling pathway, and autophagy arising from inhibition of Rac3. Co-immunoprecipitation experiments were performed to investigate the interaction between Rac3 and proteins related to endoplasmic reticulum stress. Co-immunoprecipitation was performed to investigate the structural domains between Rac3 and HSPA5.
RESULTS: The expression of ATF4 and DDIT4 was upregulated, which inhibited the MTOR signaling pathway and induced autophagy of human non-small cell lung cancer cells after Rac3 siRNA was introduced. The degree of acetylation of the substrate, HSPA5, increased and the endoplasmic reticulum stress response was activated after Rac3 was inhibited.
CONCLUSION: In conclusion, the degree of acetylation of HSPA5 increased and it was dissociated from the receptor, EIF2AK3, on the endoplasmic reticulum membrane, thus causing the endoplasmic reticulum stress response. Endoplasmic reticulum stress activated the expression of the ATF4 protein, upregulated the level of DDIT4, inhibited the MTOR signaling pathway, and caused cellular autophagy.

Lung cancer is still the leading cause of malignant deaths in the world. It is of great importance to find novel functional genes for the tumorigenesis of lung cancer. We demonstrated that Rac3 could promote cell proliferation and inhibit apoptosis in lung adenocarcinoma cell line A549 previously. The aim of this study was to investigate the function and mechanism of Rac3 in lung adenocarcinoma cell lines. Immunohistochemistry staining was performed in 107 lung adenocarcinoma tissues and matched non-tumor tissues. Multivariate analysis and Kaplan-Meier analysis were used to investigate the correlation between Rac3 expression and the clinical outcomes. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony formation assay, and flow cytometry analysis were employed to determine the proliferative ability, cell cycle distribution, and apoptosis in H1299 and H1975 cell lines. Gene expression microarray and pathway analysis between the Rac3-siRNA group and the control group in A549 cells were performed to investigate the pathways and mechanism of Rac3 regulation. Rac3 was shown to be positively expressed in lung adenocarcinoma tissues, and the expression of Rac3 associates with longer survival in lung adenocarcinoma patients. Silencing of Rac3 significantly induced cell growth inhibition, colony formation decrease, cell cycle arrest, and apoptosis of lung adenocarcinoma cell lines, which accompanied by obvious downregulation of CCND1, MYC, and TFDP1 of cell cycle pathway involving in the tumorigenesis of lung adenocarcinoma based on the gene expression microarray. In conclusion, these findings suggest that Rac3 has the potential of being a therapeutic target for lung adenocarcinoma.

Ras homology GTPase activation protein 6 (Arhgap6), as a member of the rhoGAP family of proteins, performs vital functions on the regulation of actin polymerization at the plasma membrane during several cellular processes. The role of Arhgap6 in the progression and development of cancer remains nearly unknown. This study aimed at exploring the effects of Arhgap6 on cervical carcinoma. Human cervical cancer cells HeLa and SiHa were transduced with a lentivirus targeting Arhgap6 (Arhgap6+), while CaSki and C4-1 cells were transfected with miRNA. Cell proliferation was identified by Cell Counting Kit-8 (CCK-8). Cell cycle distribution and cell apoptosis were identified by flow cytometry. The capacity of cell migration, invasion, and adhesion were detected by Transwell assay. Further, quantitative real-time PCR (qRT-PCR) and western blot were used to analyze the expression levels of Arhgap6 and several tumor-related genes. Co-immunoprecipitation assay was performed to validate the interaction between Arhgap6 and Rac3 (Ras-related C3 botulinum toxin substrate 3). Results showed that Arhgap6 inhibited cell proliferation, migration, invasion, and adhesion of cervical carcinoma, induced cell apoptosis, and caused cell cycle arrest in the G0/G1 phase (n = 3, p < 0.05). Expression of the tumor suppressor genes and oncogenes were up- and down-regulated respectively by Arhgap6, and Rac3 was proved to be the target of Arhgap6. Besides, in in vivo assays, tumor size and weight were destructed in Arhgap6+ athymic nude mouse. This study indicated that Arhgap6 may play a role in the treatment of cervical cancer as a tumor supressor.

BACKGROUND: Rac3, a member of the Rac family of small guanosine triphosphatases (GTPases), regulates a variety of cell functions, including the organization of the cytoskeleton, cell migration, and invasion. Overexpression of Rac3 has been reported in several human cancers. However, the role of Rac3 in lung cancer (LC) has not been determined in detail. The purpose of this study was to investigate the effect of silencing of Rac3 expression in human LC cells and the consequences for cell survival.
MATERIALS AND METHODS: Lentivirus small hairpin RNA (shRNA) interference techniques were utilized to knock down the Rac3 gene. Gene and protein expression was quantified by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. LC cell apoptosis was examined by annexin V-APC /propidium iodide staining.
RESULTS: Efficient silencing of Rac3 strongly inhibited A549 cell proliferation and colony formation ability, and significantly decreased tumor growth. Moreover, flow cytometry analysis showed that knockdown of Rac3 led to G2/M phase cell cycle arrest as well as an excess accumulation of cells in the G1 and S phase.
CONCLUSIONS: Thus, functional analysis using shRNAs revealed a critical role for Rac3 in the tumor growth of LC cells. shRNA silencing of Rac3 could provide an effective strategy to treat LC.

Gene expression microarrays are widely used to investigate molecular targets in cancers, including lung cancer. In this study, we analyzed online non-small cell lung cancer (NSCLC) microarray databases, to screen the key genes and pathways related to NSCLC by bioinformatics analyses. And then, the expression levels of two selected genes in the down-regulated co-pathways, myosin light chain kinase (MYLK) and myosin regulatory light chain 9 (MYL9), were determined in tumor, paired paraneoplastic, and normal lung tissues. First, gene set enrichment analysis and meta-analysis were conducted to identify key genes and pathways that contribute to NSCLC carcinogenesis. Second, using the total RNA and protein extracted from lung cancer tissues (n = 240), adjacent non-cancer tissues (n = 240), and normal lung tissues (n = 300), we examined the MYLK and MYL9 expression levels by quantitative real-time PCR and Western blot. Finally, we explored the correlations between mRNA and protein expressions of these two genes and the clinicopathological parameters of NSCLC. Fifteen up-regulated and nine down-regulated co-pathways were observed. A number of differentially expressed genes (CALM1, THBS1, CSF3, BMP2, IL6ST, MYLK, ROCK2, IL3RA, MYL9, PPP2CA, CSF2RB, CNAQ, GRIA2, IL10RA, IL10RB, IL11RA, LIFR, PLCB4, and RAC3) were identified (P < 0.01) in the down-regulated co-pathways. The expression levels of MYLK and MYL9, which act downstream of the vascular smooth muscle contraction signal pathway and focal adhesion pathway, were significantly lower in cancer tissue than those in the paraneoplastic and normal tissues (P < 0.05). Moreover, the expression levels of these two genes in stages III and IV NSCLC were significantly increased, when compared to stages I and II, and expressions levels in NSCLC with lymphatic metastasis were higher than that without lymphatic metastasis (P < 0.05). Additionally, significant lower expression levels of the two genes were found in smokers than in nonsmokers (P < 0.05). In contrast, gender, differentiated degrees, and pathohistological type appeared to have no impact on these gene expressions (P > 0.05). These findings suggested that low MYLK and MYL9 expressions might be associated with the development of NSCLC. These genes may be also relevant to NSCLC metastasis. Future investigations with large sample sizes needed to verify these findings.

BACKGROUND: Rac3 is a small GTPase multifunctional protein that regulates cell adhesion, migration, and differentiation. It has been considered as an oncogene in breast cancer; however, its role in esophageal cancer and the regulation of its stability have not been studied. F-box proteins are major subunits within the Skp1-Cullin-1-F-box (SCF) E3 ubiquitin ligases that recognize particular substrates for ubiquitination and proteasomal degradation. Recently, we have shown that SCFFBXL19 targets Rac1 and RhoA, thus regulating Rac1 and RhoA ubiquitination and degradation. Here, we demonstrate the role of FBXL19 in the regulation of Rac3 site-specific ubiquitination and stability. Expression of TGFβ1 is associated with poor prognosis of esophageal cancer. TGFβ1 reduces tumor suppressor, E-cadherin, expression in various epithelial-derived cancers. Here we investigate the role of FBXL19-mediated Rac3 degradation in TGFβ1-induced E-cadherin down-regulation in esophageal cancer cells.
METHODS: FBXL19-regulated endogenous and over-expressed Rac3 stability were determined by immunoblotting and co-immunoprecipitation. Esophageal cancer cells (OE19 and OE33) were used to investigate TGFβ1-induced E-cadherin down-regulation by Immunoblotting and Immunostaining.
RESULTS: Overexpression of FBXL19 decreased endogenous and over-expressed Rac3 expression by interacting and polyubiquitinating Rac3, while down-regulation of FBXL19 suppressed Rac3 degradation. Lysine166 within Rac3 was identified as an ubiquitination acceptor site. The FBXL19 variant with truncation at the N-terminus resulted in an increase in Rac3 degradation; however, the FBXL19 variant with truncation at the C-terminus lost its ability to interact with Rac3 and ubiquitinate Rac3 protein. Further, we found that Rac3 plays a critical role in TGFβ1-induced E-cadherin down-regulation in esophageal cancer cells. Over-expression of FBXL19 attenuated TGFβ1-induced E-cadherin down-regulation and esophageal cancer cells elongation phenotype.
CONCLUSIONS: Collectively these data unveil that FBXL19 functions as an antagonist of Rac3 by regulating its stability and regulates the TGFβ1-induced E-cadherin down-regulation. This study will provide a new potential therapeutic strategy to regulate TGFβ1 signaling, thus suppressing esophageal tumorigenesis.

RAC3 is an oncogene naturally overexpressed in several tumors. Besides its role as coactivator, it can exert several protumoral cytoplasmic actions. Autophagy was found to act either as a tumor suppressor during the early stages of tumor development, or as a protector of the tumor cell in later stages under hypoxic conditions. We found that RAC3 overexpression inhibits autophagy when induced by starvation or rapamycin and involves RAC3 nuclear translocation-dependent and -independent mechanisms. Moreover, hypoxia inhibits the RAC3 gene expression leading to the autophagy process, allowing tumor cells to survive until angiogenesis occurs. The interplay between RAC3, hypoxia, and autophagy could be an important mechanism for tumor progression and a good target for a future anticancer therapy.

Estrogen receptor alpha (ERα) is a ligand-dependent nuclear receptor that is important in breast cancer genesis, behavior and response to hormone-based therapies. A T7 phage display screen against full-length human ERα, coupled with genome-wide exon arrays, was used to identify RAC3 as a putative ERα co-regulator. RAC3 is a Rho family small GTPase that is associated with cytoskeletal rearrangement. We demonstrate a novel role for nuclear RAC3 as an ERα transcriptional activator, with prognostic implications for metastatic disease. Through in vitro and cell-based studies, RAC3 was shown to exist in a GTP-bound state and act as a ligand specific ERα co-activator of E2-induced transcription. Overexpression of RAC3 induced pro-growth and pro-migratory genes that resulted in increased migration of ERα-positive breast cancer cells. Chemical inhibition and genetic knockdown of RAC3 antagonized E2-induced cell proliferation, cell migration and ERα mediated gene expression, indicating that RAC3 is necessary for full ERα transcriptional activity. In agreement with the molecular and cellular data, RAC3 overexpression in ERα-positive breast cancers correlated with a significant decrease in recurrence free survival and a significant increase in the odds ratio of metastasis. In conclusion, RAC3 is a novel ERα co-activator that promotes cell migration and has prognostic value for ERα-positive breast cancer metastasis. RAC3 may also be a useful therapeutic target for ERα-positive breast cancers.

The androgen receptor (AR) is a ligand activated nuclear receptor, which regulates transcription and stimulates growth of androgen dependent prostate cancer. To regulate transcription, AR recruits a series of coactivators that modify chromatin and facilitate transcription. However, information on ligand and target gene-specific requirements for coactivators is limited. We compared the actions of the p160 coactivators SRC-1 and SRC-3/RAC3 with SRA (steroid receptor RNA activator). All three coactivate AR in the presence of agonist as expected. However, overexpression of either SRC-1 or SRC-3 increased AR activity in response to the partial antagonist, cyproterone acetate, whereas SRA was unable to stimulate AR activity under these conditions. Using siRNA to reduce expression of these coactivators in LNCaP cells, we also found promoter specific requirement for these coactivators. SRC-3 is required for optimal androgen dependent induction of PSA, TMPRSS2, and PMEPA1 whereas SRA is required only for optimal induction of the TMPRSS2 gene. These data indicate that different groups of AR target genes have distinct requirements for coactivators and response to AR ligands.

The Rho GDP dissociation inhibitor D4-GDI is overexpressed in some human breast cancer cell lines (Zhang, Y., and Zhang, B. (2006) Cancer Res. 66, 5592-5598). Here, we show that silencing of D4-GDI by RNA interference abrogates tumor growth and lung metastasis of otherwise highly invasive MDA-MB-231 breast cancer cells. Under anchorage-independent culture conditions, D4-GDI-depleted cells undergo rapid apoptosis (anoikis), which is known to hinder metastasis. We also found that D4-GDI associates with Rac1 and Rac3 in breast cancer cells, but not with other Rho GTPases tested (Cdc42, RhoA, RhoC, and TC10). Silencing of D4-GDI results in constitutive Rac1 activation and translocation from the cytosol to cellular membrane compartments and in sustained activation of p38 and JNK kinases. Rac1 blockade inhibits p38/JNK kinase activities and the spontaneous anoikis of D4-GDI knockdown cells. These results suggest that D4-GDI regulates cell function by interacting primarily with Rac GTPases and may play an integral role in breast cancer tumorigenesis. D4-GDI could prove to be a potential new target for therapeutic intervention.

Bone marrow engraftment in the context of hematopoietic stem cell and progenitor (HSC/P) transplantation is based on the ability of intravenously administered cells to lodge in the medullary cavity and be retained in the appropriate marrow space, a process referred to as homing. It is likely that homing is a multistep process, encompassing a sequence of highly regulated events that mimic the migration of leukocytes to inflammatory sites. In leukocyte biology, this process includes an initial phase of tethering and rolling of cells to the endothelium via E- and P-selectins, firm adhesion to the vessel wall via integrins that appear to be activated in an "inside-out" fashion, transendothelial migration, and chemotaxis through the extracellular matrix (ECM) to the inflammatory nidus. For HSC/P, the cells appear to migrate to the endosteal space of the bone marrow. A second phase of engraftment involves the subsequent interaction of specific HSC/P surface receptors, such as alpha(4)beta(1) integrin receptors with vascular cell-cell adhesion molecule-1 and fibronectin in the ECM, and interactions with growth factors that are soluble, membrane, or matrix bound. We have utilized knockout and conditional knockout mouse lines generated by gene targeting to study the role of Rac1 and Rac2 in blood cell development and function. We have determined that Rac is activated via stimulation of CXCR4 by SDF-1, by adhesion via beta(1) integrins, and via stimulation of c-kit by the stem cell factor-all of which involved in stem cell engraftment. Thus Rac proteins are key molecular switches of HSC/P engraftment and marrow retention. We have defined Rac proteins as key regulators of HSC/P cell function and delineated key unique and overlapping functions of these two highly related GTPases in a variety of primary hematopoietic cell lineages in vitro and in vivo. Further, we have begun to define the mechanisms by which each GTPase leads to specific functions in these cells. These studies have led to important new understanding of stem cell bone marrow retention and trafficking in the peripheral circulation and to the development of a novel small molecule inhibitor that can modulate stem cell functions, including adhesion, mobilization, and proliferation. This chapter describes the biochemical footprint of stem cell engraftment and marrow retention related to Rho GTPases. In addition, it reviews abnormalities of Rho GTPases implicated in human immunohematopoietic diseases and in leukemia/lymphoma.

To investigate the molecular effects of growth factor independence 1B (Gfi-1B), a transcription factor essential for the development of hematopoietic cells and differentiation of erythroid and megakaryocytic lineages, the naturally Gfi-1B overexpressing cell line K562 was cultured in the presence of Gfi-1B target-specific small interfering RNA (siRNA). SiRNA treatment significantly knocked down Gfi-1B expression with an efficiency of nearly 90%. Analysis of the siRNA silencing protocol by colony-forming units ensured that it was not cytotoxic. Samples from Gfi-1B overexpressing cells and cells with knocked-down Gfi-1B were analyzed by oligonucleotide microarray technology and based upon rigorous statistical analysis of the data; relevant genes were chosen for confirmation by reserve transcriptase-polymerase chain reaction, including MYC/MYCBP and CDKN1A. Interestingly, transcripts within components of the signalling cascade of immune cells (PLD1, LAMP1, HSP90, IL6ST), of the tyrosine kinase pathway (TPR, RAC3) and of the transcription factors (RAC3, CEP290, JEM-1, ATR, MYC, SMC3, RARA, RBBP6) were found to be differentially expressed in Gfi-1B overexpressing cells compared to controls. Individual genes such as ZDHHC17, DMXL1, ZNF292 were found to be upregulated in Gfi-1B overexpressing cells. In addition, down-regulated transcripts showed cell signaling transcripts for several chemokine gene members including GNAL, CXCL5, GNL3L, GPR65, TMEM30, BCL11B and transcription factors (GTF2H3, ATXN3). In conclusion, several essential cell signalling factors, as well as transcriptional and post-translational regulation genes were differentially expressed in cells that overexpressed Gfi-1B compared to control cells with knocked-down Gfi-1B. Our data indicate that Gfi-1B signalling is important for commitment and maturation of hematopoietic cell populations.

Estrogen is recognized as a significant modifier in the development, growth and invasion of uterine endometrial cancer. Steroid receptor coactivator-3 (SRC-3; AIB1, ACTR, RAC3, TRAM-1, and pCIP) is a member of the p160 family of coactivator for nuclear hormone receptors including estrogen receptor (ER). It is reported that SRC-3 is overexpressed in various cancers. However, SRC-3 expression manner in uterine endometrial cancer is not fully understood. In this study, we showed SRC-3 mRNA expression correlates with clinical stage, depth of myometrial invasion and dedifferentiation. The prognosis of the 25 patients with higher expression of SRC-3 mRNA in uterine endometrial cancers was extremely poor (36%), whereas the 24-month survival rate of the 15 patients with lower expression of SRC-3 mRNA was 96%. These data indicate that SRC-3 might be an important indicator of uterine endometrial cancer advancement and survival.

Endocrine therapy for advanced prostate cancer is based on androgen ablation or blockade of the androgen receptor (AR). AR action in prostate cancer has been investigated in a number of cell lines, their derivatives, and transgenic animals. AR expression is heterogenous in prostate cancer in vivo; it could be detected in most primary tumors and their metastases. However, some cells lack the AR because of epigenetic changes in the gene promoter. AR expression increases after chronic androgen ablation in vitro. In several xenografts, AR upregulation is the most consistent change identified during progression towards therapy resistance. In contrast, the AR pathway may be by-passed during chronic treatment with a nonsteroidal anti-androgen. AR sensitivity in prostate cancer increases as a result of activation of the Ras/mitogen-activated protein kinase pathway. One of the major difficulties in endocrine therapy for prostate cancer is acquisition of agonistic properties of AR antagonists observed in the presence of mutated AR. Enhancement of AR function by associated coactivator proteins has been extensively investigated. Cofactors SRC-1, RAC3, p300/CBP, TIF-2, and Tip60 are upregulated in advanced prostate cancer. Most studies on ligand-independent activation of the AR are focused on Her-2/neu and interleukin-6 (IL-6). On the basis of studies that showed overexpression and activation of the AR in advanced prostate cancer, it was suggested that novel therapies that reduce AR expression will provide a benefit to patients. There is experimental evidence showing that prostate tumor growth in vitro and in vivo is inhibited following administration of chemopreventive drugs or antisense oligonucleotides that downregulate AR mRNA and protein expression.

Steroid receptor coactivator-3 (SRC-3, also known as NCoA3, AIB1, p/CIP, RAC3, ACTR, and TRAM1), localized on a frequently amplified region, 20q12, has been associated with multiple cancers, including breast, gastric and prostate cancers. Although SRC-3 has been implicated as an oncogene, compelling evidence has only recently emerged implicating it as a causal factor in the genesis of human cancers. Here, we summarize recent evidence that indicates aberrant SRC-3 expression is important in hormone-sensitive and -insensitive human cancers.

It has been suggested that the steroid receptor coactivatior-3 (SRC-3) gene, also known as AIB1, ACTR, RAC3, p/CIP and TRAM-1, located at 20q12, plays an oncogenic role in several types of human cancers. In this study, we examined the encoded protein expression of SRC-3 and its copy number in 221 human esophageal squamous cell carcinomas (ESCCs). In this ESCC series, the overexpression and increased copy number of SRC-3 gene was detected in 46 and 13% of ESCCs, respectively. In addition, overexpression of SRC-3 was observed more frequently in primary ESCCs in late T stages (T3/T4) than that in earlier T1/T2 stages (P<0.05), but no significant association of expression of SRC-3 and status of lymph node metastases was observed (P>0.05). These results suggest that overexpression of SRC-3, caused by gene amplification/gain or other molecular mechanisms, might provide a selective advantage for the development and local invasion of certain subsets of ESCC. In addition, a significant correlation (P<0.05) of overexpression of SRC-3 with increased cell proliferation (through detection of Ki-67 expression) was observed in these ESCCs. These findings suggest a potential role of SRC-3 in the control of ESCC cell proliferation; such may be responsible, at least in part, for tumorigenesis and/or progression of ESCC.

INTRODUCTION: The metastatic progression of cancer is a direct result of the disregulation of numerous cellular signaling pathways, including those associated with adhesion, migration, and invasion. Members of the Rac family of small GTPases are known to act as regulators of actin cytoskeletal structures and strongly influence the cellular processes of integrin-mediated adhesion and migration. Even though hyperactivated Rac proteins have been shown to influence metastatic processes, these proteins have never been directly linked to metastatic progression.
METHODS: To investigate a role for Rac and Cdc42 in metastatic breast cancer cell invasion and migration, relative endogenous Rac or Cdc42 activity was determined in a panel of metastatic variants of the MDA-MB-435 metastatic human breast cancer cell line using a p21-binding domain-PAK pull down assay. To investigate the migratory and invasive potential of the Rac isoforms in human breast cancer, namely Rac1 and the subsequently cloned Rac3, we stably expressed either dominant active Rac1 or dominant active Rac3 into the least metastatic cell variant. Dominant negative Rac1 or dominant negative Rac3 were stably expressed in the most metastatic cell variant. Cell lines expressing mutant Rac1 or Rac3 were analyzed using in vitro adhesion, migration and invasion assays.
RESULTS: We show that increased activation of Rac proteins directly correlates with increasing metastatic potential in a panel of cell variants derived from a single metastatic breast cancer cell line (MDA-MB-435). The same correlation could not be found with activated Cdc42. Expression of a dominant active Rac1 or a dominant active Rac3 resulted in a more invasive and motile phenotype. Moreover, expression of either dominant negative Rac1 or dominant negative Rac3 into the most metastatic cell variant resulted in decreased invasive and motile properties.
CONCLUSION: This study correlates endogenous Rac activity with high metastatic potential and implicates Rac in the regulation of cell migration and invasion in metastatic breast cancer cells. Taken together, these results suggest a role for both the Rac1 and Rac3 GTPases in human breast cancer progression.

BACKGROUND AND PURPOSE: Androgen receptor (AR) coactivators modulate the transcriptional activity of AR and may activate AR-associated genes upon androgen deprivation. Changes of expression patterns may help reveal the role of AR cofactors in androgen-independent prostate cancer. This study investigated changes of expression patterns of several AR coactivators after androgen deprivation in an androgen-dependent prostate cancer cell line (LNCaP).
METHODS: LNCaP cells were cultured in RPMI medium with charcoal/dextran-treated fetal bovine serum for 28 days. Samples of total RNA collected at 1-week intervals were analyzed using a non-isotopic multi-probe ribonuclease protection assay system with a human AR multi-probe template set. Expression changes of 10 mRNAs of interest (AR, Rb, ARA160, ARA24, ARA54, ARA55, ARA70, BRCA1, F-SRC-1, and RAC3) were analyzed simultaneously as AR and AR-associated cofactors in 1 hybridization reaction.
RESULTS: Seven of 10 cofactors tested were expressed in LNCaP cells. Only Rb, ARA55 and BRCA1 were not detected. LNCaP cells cultured in charcoal-treated serum for 28 days clearly showed more than 1.5-fold increases in AR, ARA160, and ARA70 expression, while expression of ARA24 and ARA54 increased less than 1.5-fold. Only RAC3 and F-SRC-1 decreased in RNA expression. Cell morphology features changed gradually into neuron-like shapes with elongated cytoplasm during the culture period. Cell growth almost ceased 28 days after the start of culture.
CONCLUSION: The changes in expression pattern of AR and AR cofactors and in particular the remarkable increase in AR, ARA160 and ARA70 support the role of AR cofactors in modulating prostate cancer cell growth from androgen dependence to androgen independence.

Rac3 may play an important role in tumor growth but little is known about its expression and mutation in human tumor tissues. We examined the expression of Rac3 using RT-PCR and mutation of the Rac3 gene by DNA sequencing. Overexpression of the Rac3 gene occurred in 19% (5/26) of brain tumors; 3 of 9 (33%) meningiomas, 1 of 11 (9%) astrocytomas and 1 of 6 (17%) pituitary adenomas. Two of the 3 meningiomas with Rac3 overexpression were recurrent meningiomas. The only astrocytoma with Rac3 overexpression was a glioblastoma multiforme. Mutation of the Rac3 gene occurred in 63% (12/19) of brain tumours; 4 of 7 (57.1%) meningiomas, 4 of 5 (80%) pituitary adenomas and 4 of 7 (57.1%) astrocytomas. Except in one astrocytoma, the other four tumors with Rac3 overexpression (3 meningiomas and one pituitary adenoma) did not have Rac3 mutations. Our data is the first report of the frequency of Rac3 overexpression and mutation in human brain tumors. Overexpression may be associated with aggressive tumor behavior. The relationship between Rac3 expression and mutation requires further investigation.

Overexpression or amplification of ACTR (also named AIB1, RAC3, p/CIP, TRAM-1, and SRC-3), a member of the p160 family of coactivators for nuclear hormone receptors, has been frequently detected in multiple types of human tumors, including breast cancer. However, its role in cancer cell proliferation and the underlying mechanism are unclear. Here, we show that overexpression of ACTR not only enhances estrogen-stimulated cell proliferation but also, more strikingly, completely negates the cell cycle arrest effect by tamoxifen and pure antiestrogens. Unexpectedly, we found that ACTR directly interacts, through its N-terminal domain, with E2F1 and is recruited to E2F target gene promoters. Elevation of ACTR in quiescent cells strongly stimulates the transcription of a subset of E2F-responsive genes that are associated with the G(1)/S transition. We also demonstrated, by adenovirus vector-mediated RNA interference, that ACTR is required for E2F1-mediated gene expression and the proliferation of estrogen receptor (ER)-negative breast cancer cells. Moreover, the ability of elevated ACTR to promote estrogen-independent cell proliferation depends on the function of E2F1 and the association between ACTR and E2F1, but not ER. Thus, our results reveal an essential role of ACTR in control of breast cancer cell proliferation and implicate the ACTR-E2F1 pathway as a novel mechanism in antiestrogen resistance.

Rho GTPases were previously shown to have an important role in cancer development and progression, including cell transformation, proliferation, invasion, metastasis, and angiogenesis. However, there is still little information available on the clinical significance of Rho GTPases expression in human cancer specimens. In the present study, we systemically investigated the mRNA expression levels of seven main members RhoA, RhoB, RhoC, Rac1, Rac2, Rac3, and Cdc42 of Rho family using semi-quantitative reverse transcription-PCR in 53 patients with gastric carcinoma and 7 gastric cancer cell lines. The total and activities of RhoA, Rac1 and Cdc42 in 5 gastric cancer cell lines were also examined. The mean mRNA expression levels of RhoA and Rac1 in gastric cancer tissue specimens were significantly higher than those in the adjacent non-tumorous tissue specimens (p < 0.01). The higher expression of RhoA was significantly correlated with higher TNM stage (p < 0.05) as well as with pooly differentiated histological type (p < 0.05) of gastric carcinoma. The increased expression of Rac1 was related to higher TNM stages of gastric carcinoma (p < 0.05). The expression levels of mRNA, total protein and activities of RhoA and Rac1 in 7 gastric cancer cell lines were all higher than that in gastric mucosal epithelial cell line GES-1. These findings indicate that RhoA and Rac1 may play important roles in the carcinogenesis and progression of gastric carcinoma.

Rac3, a novel member of the Rho subfamily of the small GTPases, is frequently activated in cultured breast cancer cells and has been shown to mediate its effect via the p21-activated kinase (Pak) pathway. In order to evaluate these findings in vivo, we generated transgenic mice that express human constitutively active V12Rac under the control of the mouse mammary tumor virus (MMTV) promoter, which targets the transgene expression to the mammary epithelium. V12Rac3 expression could be detected during the first pregnancy, and the transgenic mammary gland tissues displayed an elevated Pak1 phosphorylation. Although milk proteins, beta-casein and whey acidic protein were expressed and milk fat globules accumulated normally during pregnancy, 60% of transgenic mothers failed to nurse their pups. Surprisingly, although full lactational differentiation was never achieved in transgenic mice, gland involution was incomplete. For 5 days after weaning, involution was normal, but thereafter, epithelial islands characteristic of this early stage of involution persisted for months. The apoptotic index decreased after 5 days, and these glands were associated with increased p38 MAPK phosphorylation. Nine months postpartum, the transgenic mammary glands still demonstrated a large amount of persistent epithelial islands and abnormally large ducts with lymphocyte infiltration, whereas the tissues of non-transgenic controls had returned to their normal 'virgin-like' phenotype. These data show that sustained activation of Rac3 in the mammary epithelium leads to impaired mammary gland physiology and results in the formation of mammary gland lesions.

BACKGROUND: Obesity in women has been associated with a variety of factors, including genetic predisposition, social class, early age at menarche, exercise, alcohol consumption and diet. Obesity is a risk factor for the occurrence and the recurrence of breast cancer in postmenopausal women, perhaps because of increased exposure to estrogen, insulin and insulin-like growth factors (IGFs). The progesterone receptor (PR) and the steroid hormone receptor coactivator pCIP/ACTR/AIB1/TRAM1/RAC3 (AIB1) are hypothesized to mediate signaling crosstalk between these hormonal pathways. Polymorphisms in both genes have been described and their association with breast cancer risk reported. If genetic factors contribute to obesity, and the PR and AIB1 genes influence estrogenic, insulin and IGF pathways, then genetic patterns resulting from PR and AIB1 polymorphisms may be associated with obesity in postmenopausal women.
OBJECTIVE: We compared the PR and AIB1 genotypes of postmenopausal women with breast cancer with demographic, disease-related, reproductive, lifestyle and dietary variables in terms of the strength of their relationship with obesity (BMI> or =30 kg/m2).
SUBJECTS: A total of 301 postmenopausal women previously diagnosed with Stage I, II or IIIA breast cancer, who are enrolled in the Women's Healthy Eating and Living (WHEL) study (age: 34.5-70.8 y, BMI: 17.8-54.6 kg/m2).
MEASUREMENTS: The PR polymorphism PROGINS was identified by PCR. The length of the AIB1 polyglutamine repeat was determined by PCR and nondenaturing gel electrophoresis or DNA sequencing. BMI was obtained at the baseline clinic visit upon entry into the WHEL study. Information about date of diagnosis, stage of disease, tumor hormone receptor status and adjuvant treatment received were obtained from medical records. Reproductive, menstrual history, demographic, family history of cancer, smoking history and exercise frequency and intensity information were obtained from questionnaires. Dietary and alcohol intake data came from four 24-h telephone recalls of food intake obtained at the study entry.
RESULTS: The combined inheritance of PROGINS A1/A1 and AIB1 28/29, 28/30, 28/31, 29/29 or 29/30 (AIB1 LG) genotypes (adjusted odds ratio (OR)=2.22 (95% confidence interval 1.25-3.93)) and early age at menarche (<12 y) (adjusted OR=2.34 (1.12-4.86)) were each associated with the risk for obesity. Current use of tamoxifen (adjusted OR=0.49 (0.28-0.87)) and an alcohol intake > or =10 g/day (adjusted OR=0.28 (0.11-0.77)) were inversely associated with BMI > or =30 kg/m2.
CONCLUSION: Early age at menarche and a PROGINS A1/A1+AIB1 LG genetic pattern had comparable levels of association with obesity in this cross-sectional sample of postmenopausal women with breast cancer. Since this was a cross-sectional rather than a case-control design, the association between PROGINS and AIB1 genotype and obesity found in this sample should be considered preliminary, and must be re-evaluated with a new and larger sample.

Nuclear hormone receptors are ligand-dependent transcription factors that require coactivators to regulate target gene expression. The steroid receptor coactivator-3 (SRC-3), also known as p/CIP, RAC3, AIB1, ACTR and TRAM-1, is a cancer-amplified coactivator in the SRC gene family that also contains SRC-1 and TIF2/GRIP1. SRC-3 interacts with nuclear receptors and certain other transcription factors, recruits histone acetyltransferases and methyltransferases for chromatin remodeling and facilitates target gene transcription. Accumulated results from both ex vivo and animal model studies indicate that SRC-3 plays important roles in many biological processes involving cell proliferation, cell migration, cell differentiation, somatic growth, sexual maturation, female reproductive function, vasoprotection and breast cancer. This article summarizes our current knowledge about SRC-3 under the following topics: molecular cloning and characterization; molecular structure and functional mechanisms; SRC-3 as a molecular target of growth factors and cytokines; organization and expression of the SRC-3 gene; generation and characterization of SRC-3 knockout mice; role of SRC-3 in the vasoprotective effects of estrogen; role of SRC-3 in cell migration, proliferation and cancers.

The androgen receptor (AR), a transcription factor that mediates the action of androgens in target tissues, is expressed in nearly all prostate cancers. Carcinoma of the prostate is the most frequently diagnosed neoplasm in men in industrialized countries. Palliative treatment for non-organ-confined prostate cancer aims to down-regulate the concentration of circulating androgen or to block the transcription activation function of the AR. AR function during endocrine therapy was studied in tumor cells LNCaP subjected to long-term steroid depletion; newly generated sublines could be stimulated by lower concentrations of androgen than parental cells and showed up-regulation of AR expression and activity as well as resistance to apoptosis. Androgenic hormones regulate the expression of key cell cycle regulators, cyclin-dependent kinase 2 and 4, and that of the cell cycle inhibitor p27. Inhibition of AR expression could be achieved by potential chemopreventive agents flufenamic acid, resveratrol, quercetin, polyunsaturated fatty acids and interleukin-1beta, and by the application of AR antisense oligonucleotides. In the clinical situation, AR gene amplification and point mutations were reported in patients with metastatic disease. These mutations generate receptors which could be activated by other steroid hormones and non-steroidal antiandrogens. In the absence of androgen, the AR could be activated by various growth-promoting (growth factors, epidermal growth factor receptor-related oncogene HER-2/neu) and pleiotropic (protein kinase A activators, interleukin-6) compounds as well as by inducers of differentiation (phenylbutyrate). AR function is modulated by a number of coactivators and corepressors. The three coactivators, TIF-2, SRC-1 and RAC3, are up-regulated in relapsed prostate cancer. New experimental therapies for prostate cancer are aimed to down-regulate AR expression and to overcome difficulties which occur because of the acquisition of agonistic properties of commonly used antiandrogens.

OBJECTIVES: To investigate the expression of androgen receptor (AR) coactivators in the human prostate for a better understanding of androgen action in prostate cancer.
METHODS: Using reverse transcriptase-polymerase chain reaction, we examined the expression levels of AR coactivators (ARA55, SRC1, ARA54, TIF2, RAC3) in four prostate cancer cell lines (DU145, PC3, LNCaP, and LN-TR2), nine benign prostatic tissue samples, and 21 prostate cancer tissue specimens.
RESULTS: In the cell lines, SRC1 was expressed ubiquitously at almost equal amounts. Contrary to this, ARA55, ARA54, TIF2, and RAC3 displayed cell line-specific expression. In the LN-TR2 cells, established from LNCaP cells by long-term treatment with tumor necrosis factor-alpha, the expression levels of ARA55 and TIF2 were much higher than those in the LNCaP cells. In every prostatic tissue specimen, the expression levels of TIF2 and RAC3 were very low. The expression levels of ARA55 and SRC1 were higher in the cancer specimens with a higher grade or poor response to endocrine therapy than in those with a lower grade or good response to endocrine therapy.
CONCLUSIONS: Prostate cancer cells express AR coactivators. Long-term stimulation by tumor necrosis factor-alpha could increase ARA55 and TIF2 expression in LNCaP cells. The different expression of coactivators may contribute to the different response of prostate cancer to androgenic stimulation or endocrine therapy.

The t(5;17) variant of acute promyelocytic leukemia (APL) fuses the genes for nucleophosmin (NPM) and the retinoic acid receptor alpha (RARalpha). Two NPM-RAR molecules are expressed as a result of alternative RNA splicing. Both contain RARalpha sequences that encode the DNA binding, heterodimerization, and ligand activation domains of RARalpha. This study was designed to test the ability of these fusion proteins to act as transcriptional activators of retinoic acid responsive promoters. The NPM-RAR fusion proteins bind to retinoic acid response element sequences as either homodimers or as heterodimers with RXR. Transcription of retinoic acid-inducible promoters is activated by the fusion proteins in the presence of retinoic acid. The level of transactivation induced by the NPM-RAR fusions differs from the level of transactivation induced by wild-type RARalpha in both a promoter and cell specific fashion, and more closely parallels the pattern of activation of the PML-RAR fusion than wild-type RARalpha. In addition, NPM-RAR decreases basal transcription from some promoters and acts in a dominant-negative fashion when co-transfected with wild-type RARalpha. Both NPM-RAR and PML-RAR interact with the co-repressor protein SMRTe in a manner that is less sensitive than RARalpha to dissociation by retinoic acid. Retinoic acid induces binding of the co-activator protein RAC3. These data indicate that the NPM-RAR fusion proteins can modulate expression of retinoid-responsive genes in a positive or negative manner, depending on context of the promoter, and lend support to the hypothesis that aberrant transcriptional activation underlies the APL phenotype. (Blood. 2000;95:2683-2690)

The closely related small GTP-binding proteins Rac1, Rac2, and Rac3 are part of a larger Rho subfamily of Ras proteins. Because disruption of Ras signaling pathways is relevant to the pathogenesis of a wide variety of cancers, it is important to clearly define the structural and functional characteristics of the participating proteins and their encoding genes. Rho subfamily members are involved in a range of signal transduction pathways relevant to cell growth, differentiation, motility, and stress, and Rac proteins are now recognised as a necessary component of Ras-mediated cellular transformation. We previously mapped RAC3 to chromosome band 17q23--> q25, a region that contains a number of candidate tumour suppressor genes. Because of its oncogenic potential, we have now further refined the location of this gene. Here we confirm that RAC3 maps to chromosome band 17q25.3 and further show that it maps some distance telomeric of a well-characterised minimal breast and ovarian candidate tumour suppressor gene region, BROV. The genomic structure of RAC3, including exon and intron boundaries, is also presented.