Anti-apoptotic protein BRE/BRCC45 attenuates apoptosis through maintaining the expression of caspase inhibitor XIAP in mouse Lewis lung carcinoma D122 cells

Brain and Reproductive Organ Expressed (BRE), or BRCC45, is a death receptor-associated antiapoptotic protein, which is also involved in DNA-damage repair, and K63-specific deubiquitination. BRE overexpression attenuates both death receptor- and stress-induced apoptosis, promotes experimental tumor growth, and is associated with human hepatocellular and esophageal carcinoma. How BRE mediates its antiapoptotic function is unknown. Here we report based on the use of a mouse Lewis lung carcinoma cell line D122 that BRE has an essential role in maintaining the cellular protein level of XIAP, which is the most potent endogenous inhibitor of the caspases functioning in both extrinsic and intrinsic apoptosis. shRNA-mediated exhaustive depletion of BRE sensitized D122 cells to apoptosis induced not only by etopoxide, but also by TNF-α even in the absence of cycloheximide, which blocks the synthesis of antiapoptotic proteins by TNF-α-activated NF-κB pathway. In BRE-depleted cells, protein level of XIAP was downregulated, but not the levels of other antiapoptotic proteins, cIAP-1, 2, and cFLIP, regulated by the same NF-κB pathway. Reconstitution of BRE restored XIAP levels and increased resistance to apoptosis. XIAP mRNA level was also reduced in the BRE-depleted cells, but the level of reduction was less profound than that of the protein level. However, BRE could not delay protein turnover of XIAP. Depletion of BRE also increased tumor cell apoptosis, and decreased both local and metastatic tumor growth. Taken together, these findings indicate that BRE and its XIAP-sustaining mechanism could represent novel targets for anti-cancer therapy.     

BRE over-expression promotes growth of hepatocellular carcinoma

BRE, also known as TNFRSF1A modulator and BRCC45, is an evolutionarily highly conserved protein. It is a death receptor-associated protein in cytoplasm and a component of BRCA1/2-containing DNA repair complex in nucleus. BRE was found to have anti-apoptotic activity. Over-expression of BRE by transfection promoted survival of cell lines against apoptotic induction; whereas depletion of the protein by siRNA resulted in the opposite. In vivo anti-apoptotic activity of BRE was demonstrated by significant attenuation of Fas-induced acute fulminant hepatitis in transgenic mice expressing the human protein specifically in the liver. BRE was also implicated in tumor promotion by the accelerated tumor growth of Lewis Lung carcinoma transfected with human BRE; and by high expression of BRE specifically in the tumoral regions of human hepatocellular carcinoma (HCC). The present study was to test directly if transgenic expression of BRE in livers could promote HCC development in neonatal diethylnitrosamine model. By 8 months after tumor induction, the maximal sizes of tumor nodules of transgenic mice were significantly larger than those of the non-transgenic controls, although the numbers of tumor nodules between the two groups did not significantly differ. Importantly, as in human HCC, the mouse endogenous BRE level was up-regulated in mouse HCC nodules. These results show that BRE over-expression can indeed promote growth, though not initiation, of liver tumors. Furthermore, the common occurrence of BRE over-expression in human and mouse HCC suggests that up-regulation of BRE is functionally important in liver tumor development.     

H-2K double transfectants of tumor cells as antimetastatic cellular vaccines in heterozygous recipients. Implications for the T cell repertoire.

In this study we demonstrate that antitumor CTL repertoire restricted to a single MHC class I allele is higher in homozygous than in heterozygous mice. Consequently, transfection of two parental H-2K genes, but not of a single H-2K gene into a highly metastatic H-2K-negative tumor clone, resulted in abrogation of metastatic properties in F1 recipients. Clones of the 3LL carcinoma, which are low H-2Kb expressors, are nonimmunogenic and highly metastatic. Transfection of H-2K genes converted cells of such clones to nonmetastatic in syngeneic homozygous mice. However, in semi-syngeneic heterozygous mice, single H-2K transfectants retained their metastatic phenotype. In such heterozygous mice, i.e., in (H-2d x H-2b)F1, or in (H-2k x H-2b)F1, transfection of the two parental H-2K genes was required for complete abolishment of the metastatic phenotypes. In fact, in these heterozygous animals, even the local growth (i.e., tumorigenicity) of the double H-2K transfectants was significantly suppressed. These observations are attributed to the difference between homozygous and heterozygous mice with regard to the T cell repertoire restricted to a single H-2K-tumor-associated antigen complex. The reduced tumorigenicity and the complete abrogation of the metastatic phenotype was a function of a high immunogenic competence of the double transfectants in F1 heterozygous mice, which was significantly higher than that of single transfectants, as measured by the induction of CTL and of their precursors. Immunization of F1 mice by inactivated double transfectants conferred protection against metastasis formation by a subsequent graft of the parental D122 cells. Single transfectants were only marginally effective in conferring such protection. Applying an immunotherapy protocol, we observed that a series of vaccinations with double transfectants of animals already carrying a parental tumor reduced significantly the generation of metastasis by the otherwise highly metastatic D122 cells.     

Induction of tumor-specific cytotoxic T lymphocytes and natural killer cells by tumor cells transfected with the interleukin-2 gene

To study the antitumor effect of local production of interleukin-2 (IL-2) from tumor cells, the poorly immunogenic murine colon cancer cells, colon26, was transfected with murine IL-2 cDNA in a bovine papilloma virus vector. IL-2 gene transfectants (mIL2+colon26) did not alter their growth rate compared with parental colon26 cells in vitro, but reduced their tumorigenicity in vivo. Immunization with mIL2+colon26 cells could induce protective immunity against parental colon26 cells. Following intravenous challenges, the colonies of lung metastasis were also inhibited. Moreover, inoculation of mIL2+ colon26 cells slowed the growth of challenged renal cell carcinoma cells, RenCa. Intraperitoneal inoculation of IL-2 gene transfectants generated a large number of peritoneal exudate cells and these cells had a highly cytolytic activity against colon26 and YAC-1. These results suggest that inoculation with IL-2 transfected tumor cells can stimulate not only cytotoxic T lymphocytes but also natural killer cells, and that these cells will act as antitumor effector cells in host animals.     

Role of syndecan-1 proteoglycan in the invasiveness of HT-1080 fibrosarcoma

Syndecan-1 is a transmembrane heparan sulfate proteoglycan which plays pivotal role in cell-cell and cell-extracellular matrix interactions. However, its implication in the establishment of malignant phenotype is still controversial. Its expression indicates differentiated phenotype in certain tumors, while it confers invasive nature for others. For the better understanding of the role of syndecan-1 in cancer we transfected HT-1080 fibrosarcoma cell line with the full and a truncated construct of syndecan-1 and established stable cell lines with them. We studied the in vitro and in vivo growth capacity and metastatic potential of the transfectants in comparison with the cell line bearing only the EGFP expression vector. Our results showed that the growth rate of syndecan transfectants increased and they developed more lung metastases than the control cells. As local growth of the full transfectant was faster than that of the 78sig we presume that the full protein and maybe the shedding is needed for the local development of the tumor, but the intracellular and transmembrane domain is sufficient to promote metastasis formation.     

Identification of metastasis-associated proteins by proteomic analysis and functional exploration of interleukin-18 in metastasis

Very little is currently known about mechanisms underlying cancer metastasis. In the present study, metastasis-associated proteomes were separated and identified by comparative proteomic analysis, and the metastasis-related function of candidate protein interleukin-18 (IL-18) was further elucidated. First, a pair of highly and poorly metastatic sublines (termed PLA801D and PLA801C, respectively), originating from the same parental PLA801 cell line, was identified by spontaneous tumorigenicity and metastasis in vivo and characterized by metastatic phenotypes analysis in vitro. Subsequently, a proteomic approach was used to compare the protein expression profiles between PLA801C and PLA801D sublines. Eleven proteins were identified and further verified by one-dimensional Western blotting, Northern blot and/or semiquantitative reverse transciptase polymerase chain reaction analysis. Compared with those in poorly metastatic PLA801C subline, cytokeratin 18, tissue transglutaminase, Rho GDP-dissociation inhibitor 1, tropomyosin, fibroblast type, IL-18 and annexin I were significantly up-regulated, while protein disulfide isomerase, heat shock protein 60, peroxiredoxin 1, chlorine intracellular channel protein 1 (CLI1) and creatine kinase, B chain were significantly down-regulated in the highly metastatic PLA801D subline. Intriguingly, all the identified candidate proteins except for CLI1 have been shown to be somehow associated with distinct aspects of tumor metastasis such as cell growth, motility, invasion, adhesion, apoptosis and tumor immunity, etc. Considering that IL-18 was present in highly metastatic PLA801D but absent in poorly metastatic PLA801C, the association of IL-18 with metastasis was further elucidated by introducing IL-18 sense/IL-18 antisense into PLA801C/PLA801D sublines simultaneously. The results demonstrated that ectopically expressed IL-18 promoted cell motility in vitro and down-regulated E-cadherin expression of PLA801C transfectants, while IL-18 antisense remarkably decreased cell invasion potency in vitro and notably increased E-cadherin expression of PLA801D transfectants, indicating that IL-18 might play a role in metastasis by inhibiting E-cadherin expression.     

Growth-suppressive function of human connexin32 in a conditional immortalized mouse hepatocyte cell line

Mouse hepatocytes immortalized with a temperature-sensitive allele of the SV40 large T-antigen (CHST8 cells) were found to lack the high expression of the gap junction proteins Cx26 and Cx32 that characterizes normal mouse hepatocytes, expressing instead Cx43 and Cx45 at minimal levels. In order to examine the growth suppressive function of Cx32 on hepatocytes, we transfected these CHST8 cells with human Cx32 complementary deoxyribonucleic acid and measured the growth rates at 33, 37, and 39 degrees C. Expression of human Cx32 and its messenger ribonucleic acid in the stable cell lines was confirmed by immunocytochemistry and by Western and Northern blots analyses. Dye transfer following lucifer yellow injection into the transfectants was extensive; Cx32 channels displayed unitary conductances of about 70 pS and were moderately voltage sensitive. When cultured at 33 and 39 degrees C, growth rates of both parental cells and transfectants were of the same level. When examined at 37 degrees C, growth rate of the transfectant, which highly expressed Cx32 at the membranes, was significantly decreased compared to the parental cells. However, no changes in the expression of Cx32 protein in the transfectants were observed between 33 and 37 degrees C. These results suggest that Cx32 expression could inhibit hepatocyte growth in vitro using the conditional immortalized cells. Cx32 transfectants using a conditional immortalized mouse hepatocyte may be useful for examining the mechanisms of growth and differentiation in hepatocytes by gap junction expression.     

Recognition and prevention of tumor metastasis by the NK receptor NKp46/NCR1

NK cells employ a variety of activating receptors to kill virally infected and tumor cells. Prominent among these receptors are the natural cytotoxicity receptors (NCRs) (NKp30, NKp44, and NKp46), of which only NKp46 has a mouse ortholog (NCR1). The tumor ligand(s) of NKp46/NCR1 is still unknown, but it was shown that the human NKp46 and the mouse NCR1 are involved in tumor eradication both in vitro and in vivo. Whether any of the NK activating receptors is involved in the prevention of tumor metastasis is unknown. To address this question, we studied the activity of the NK cell receptor NKp46/NCR1 in two spontaneous metastasis models, the B16F10.9 melanoma (B16) and the Lewis lung carcinoma (D122) in the NCR1 knockout mouse that was generated by our group, in various in vitro and in vivo assays. We demonstrated that all B16 and D122 tumors, including those generated in vivo, express an unknown ligand(s) for NKp46/NCR1. We have characterized the properties of the NKp46/NCR1 ligand(s) and demonstrated that NKp46/NCR1 is directly involved in the killing of B16 and D122 cells. Importantly, we showed in vivo that NKp46/NCR1 plays an important role in controlling B16 and D122 metastasis. Thus, to our knowledge, in this study we provide the first evidence for the direct involvement of a specific NK killer receptor in preventing tumor metastasis.     

Gicerin,an Ig-superfamily cell adhesion molecule,promotes the invasive and metastatic activities of a mouse fibroblast cell line

Gicerin is a cell adhesion molecule in the immunoglobulin (Ig) superfamily and plays an important role during development through its adhesive properties. Gicerin has two isoforms that differ in their cytoplasmic domains; s-gicerin is the shorter and l-gicerin the longer form of the protein. Gicerin is over-expressed in some sporadic tumors as well as in developing tissues. To provide direct evidence that gicerin has the potential to participate in malignant aspects of tumor cell behavior, a gicerin cDNA was introduced into L-929 cells, an endogenous gicerin-negative mouse fibroblast and subsequently analyzed for changes in their invasive and metastatic potential by implantation into nude mice and chick embryos. Compared with parental cells, both gicerin isoform transfectants showed an enhanced cell growth and invaded deeply into surrounding tissues from implanted sites in both animal models. Furthermore, l-gicerin transfectants markedly enhanced metastasis to the lung. These findings suggest that gicerin promotes the tumor growth and invasion, and the isoform bearing the longer cytoplasmic domain may play a role in metastasis.     

EphA3基因稳定表达细胞模型的建立与分析

目的：建立稳定表达外源EphA3基因的小鼠成纤维细胞株模型,初步探讨EphA3基因表达对肿瘤发生、发展的影响。方法：通过脂质体介导的方法,将真核表达载体pcDNA3.1（-）/myc-his-EphA3转染NIH3T3细胞,用Western印迹确定外源EphA3基因表达;通过MTT实验、软琼脂集落形成实验,观察EphA3基因表达对NIH3T3细胞生物学特性的影响。结果：建立了稳定转染EphA3基因的NIH3T3细胞株;EphA3基因表达的小鼠成纤维NIH3T3细胞生长速度没有明显变化,但在软琼脂上锚着非依赖生长的能力加强。结论：建立了稳定表达外源EphA3基因的NIH3T3细胞株,EphA3基因稳定表达具有诱导正常NIH3T3细胞发生恶性转化的重要生物功能。     

Herpesvirus Saimiri Transforms Human T-Cell Clones to Stable Growth without Inducing Resistance to Apoptosis

Herpesvirus saimiri (HVS) transforms human T cells to stable growth in vitro. Since HVS codes for two different antiapoptotic proteins, growth transformation by HVS might be expected to confer resistance to apoptosis. We found that the expression of both viral antiapoptotic genes was restricted to cultures with viral replication and absent in growth-transformed human T cells. A comparative examination of HVS-transformed T-cell clones and their native parental clones revealed that the expression of Bcl-2, Bcl-X_L, Bax, and members of the tumor necrosis factor receptor (TNF-R) superfamily with a death domain, namely, TNF-RI, CD95, and TRAMP, were not modulated by HVS. Expression of CD30 was induced in HVS-transformed T cells, and these cells also expressed the CD30 ligand. Uninfected and transformed T cells were sensitive to CD95 ligation but resistant to apoptosis mediated by TRAIL or soluble TNF-α. CD95 ligand was constitutively expressed on transformed but not uninfected parental T cells. Both cell types showed similar sensitivity to cell death induction or inhibition of T-cell activation mediated by irradiation, oxygen radicals, dexamethasone, cyclosporine, and prostaglandin E₂. Altogether, this study strongly suggests that growth transformation by HVS is based not on resistance to apoptosis but, rather, on utilization of normal cellular activation pathways.     

Bcl-2-mediated cell survival promotes metastasis of EpH4 betaMEKDD mammary epithelial cells

The majority of patients who succumb to cancer die from metastatic disease progression rather than from the primary tumor. Elucidation of the mechanisms underlying tissue-specific metastasis is essential to the development of effective therapies. The mitogen-activated protein kinase kinase (MEK) pathway is frequently activated in human tumors and has been shown to regulate genes involved in proliferation, migration, and invasion. Studies with MEK-transformed EpH4 mouse mammary epithelial cells showed that these cells are highly tumorigenic but have a limited metastatic ability. Detachment of epithelial cells from the extracellular matrix causes disruption of the actin cytoskeleton and induces apoptosis. Several metastatic breast carcinoma cell lines have been shown to be resistant to cell death following actin disruption. This death-resistant phenotype can be modeled by overexpressing the antiapoptotic Bcl-2 protein in cells. This suggests that mechanisms that regulate survival of extravasated tumor cells may enhance metastatic efficiency. Therefore, we examined whether expression of Bcl-2 in MEK-transformed EpH4 mammary epithelial cells could provide a survival advantage and promote metastasis. Expression of Bcl-2 in parental EpH4 mammary epithelial cells or MEK-transformed cells was insufficient to induce increased migration, invasion, or tumor development. However, Bcl-2 expression markedly enhanced spontaneous lung metastasis from orthotopically implanted primary tumors. These results clearly show that mechanisms that regulate primary tumor development are distinct from those that promote metastasis and that assays designed to isolate genes involved in transformation may fail to identify genes that are critical regulators of metastasis.     

Expression of the androgen-dependent MMTV-specific orf gene in Shionogi 115 mouse mammary tumor cells

The Shionogi 115 (S115) mouse mammary tumor cells express the MMTV-specific 1.7 kb mRNA (orf) at a high level in the presence of androgens. In lymphoid cells the orf-gene encodes a superantigen which has an important role in establishing self-tolerance but in mammary and breast cancer cells the function of the orf gene is unclear. In the present work we studied the expression of the S115 mammary tumor cell orf sequence and its role in the androgen regulated growth of S115 cells. The cloning and sequencing of the cDNA specific for the 1.7 kb mRNA from the S115 mouse mammary tumor cells revealed a 990 bp DNA sequence with a 99.8% homology to the Mtv-17 proviral strain. There was a difference of only one amino acid (isoleu-tyr) in the coding region. A peptide was synthesized according to the hypervariable C-terminal part of the predicted protein and used to raise a rabbit antiserum. The anti-S115-orf antiserum immunoprecipitated an approximately 45 kDa protein from the metabolically labeled S115 cell lysates. In order to analyze the putative functions of the protein, the orf-sequence was linked to MoMLV-LTR and to the human ß-actin promoter in the mammalian expression vectors pLTRpoly and pHßAPr-1-neo, respectively, and transfected into NIH3T3 and S115 cells. NIH3T3 transfectants expressing orf mRNA did not show a transformed phenotype in vitro. The S115 orf transfectants proliferated somewhat more slowly than the vector transfected control cells in cell culture, both in the presence or absence of androgen, but there was no obvious change in the phenotype of S115 cells or in expression of the fibroblast growth factor 8 (FGF-8). This factor is activated by Mtv-6 integration and mediates androgen effects in these cells. Unexpectedly, however, the formation of tumors by S115 orf cells in nude mice was considerably prolonged and tumor growth retarded when compared with vector transfected control or parent S115 cells. The results suggest that MMTV-orf can be functional in breast cancer cells but the mechanism of the growth repressive effect in mammary tumor remains to be analyzed.     

Suppression of human melanoma metastasis by the metastasis suppressor gene, BRMS1

We recently identified a novel metastasis suppressor gene, BRMS1, in breast cancer. Since the BRMS1 gene maps to chromosome 11q13.1-q13.2 and since chromosome 11q defects have been described in various stages of human melanoma progression, we hypothesized that BRMS1 may function as a tumor or metastasis suppressor in melanomas as well. Quantitative real-time RT-PCR revealed that BRMS1 mRNA expression was high in melanocytes, considerably reduced in early melanoma-derived cell lines, and barely detectable in advanced/metastatic cell lines. Stable transfectants of BRMS1 in the human melanoma cell lines MelJuSo and C8161.9 did not alter the tumorigenicity of either cell line, but significantly suppressed metastasis compared to vector-only transfectants. Orthotopic tumors continued to express BRMS1, but expression was lost in lung metastases. In vitro morphology, growth rate, and histology of BRMS1 transfectants were similar to controls. BRMS1 transfectants were less invasive in a collagen sandwich assay and had restored homotypic gap junctional intercellular communication (GJIC). Thus, BRMS1 functions as a metastasis suppressor in more than one tumor type (i.e., breast carcinoma and cutaneous melanoma) by modifying several metastasis-associated phenotypes.     

A death receptor-associated anti-apoptotic protein, BRE, inhibits mitochondrial apoptotic pathway

BRE, brain and reproductive organ-expressed protein, was found previously to bind the intracellular juxtamembrane domain of a ubiquitous death receptor, tumor necrosis factor receptor 1 (TNF-R1), and to down-regulate TNF-alpha-induced activation of NF-kappaB. Here we show that BRE also binds to another death receptor, Fas, and upon overexpression conferred resistance to apoptosis induced by TNF-alpha, anti-Fas agonist antibody, cycloheximide, and a variety of stress-related stimuli. However, down-regulation of the endogenous BRE by small interfering RNA increased apoptosis to TNF-alpha, but nottoetoposide, indicating that the physiological antiapoptotic role of this protein is specific to death receptor-mediated apoptosis. We further demonstrate that BRE mediates antiapoptosis by inhibiting the mitochondrial apoptotic machinery but without translocation to the mitochondria or nucleus or down-regulation of the cellular level of truncated Bid. Dissociation of BRE rapidly from TNF-R1, but not from Fas, upon receptor ligation suggests that this protein interacts with the death inducing signaling complex during apoptotic induction. Increased association of BREwith phosphorylated, sumoylated, and ubiquitinated proteins after death receptor stimulation was also detected. We conclude that in contrast to the truncated Bid that integrates mitochondrial apoptosis to death receptor-triggered apoptotic cascade, BRE inhibits the integration. We propose that BRE inhibits, by ubiquitination-like activity, components in or proximal to the death-inducing signaling complexes that are necessary for activation of the mitochondria.     

Comparative proteomic analysis reveals a function of the novel death receptor-associated protein BRE in the regulation of prohibitin and p53 expression and proliferation

The brain and reproductive organ expressed (BRE) gene encodes a highly conserved stress-modulating protein. To gain further insight into the function of this gene, we used comparative proteomics to investigate the protein profiles of C2C12 and D122 cells resulting from small interfering RNA (siRNA)-mediated silencing as well as overexpression of BRE. Silencing of BRE in C2C12 cells, using siRNA, resulted in up-regulated Akt-3 and carbonic anhydrase III expression, while the 26S proteasome regulatory subunit S14 and prohibitin were down-regulated. Prohibitin is a potential tumour suppressor gene, which can directly interact with p53. We found that cell proliferation was significantly increased after knockdown of BRE, concomitant with reduced p53 and prohibitin expression. In contrast, we observed decreased proliferation and up-regulation of p53 and prohibitin when BRE was overexpressed in the D122 cell line. In total, five proteins were found to be up-regulated after BRE over-expression. The majority of these proteins can target or crosstalk with NF-kappaB, which plays a central role in regulating cell proliferation, differentiation and survival. Our results establish a crucial role for BRE in the regulation of key proteins of the cellular stress-response machinery and provide an explanation for the multifunctional nature of BRE.     

Gene transfer of baculoviral p35 by adenoviral vector protects human cerebral neurons from apoptosis

Apoptosis plays an important role in neuronal cell death in both chronic and acute human neurological diseases, including ALS, Huntington's disease, cerebral ischemia, and HIV encephalopathy. We evaluated the ability of an extremely powerful antiapoptotic agent, baculoviral p35, to prevent apoptosis and cell death of human cerebral neurons that undergo severe neurotoxic changes in a culture system when treated with agents that are implicated in human neurological disorders, that is, tumor necrosis factor (TNFalpha) and the HIV proteins Tat and gp120. P35 is a potent broad-spectrum antiapoptotic protein derived from baculovirus, that inhibits nearly all caspases, and has other antiapoptotic actions as well. Adenoviral vectors expressing p35 (Ad. p35) or a control gene (lacZ) efficiently transduced human neurons. Treatment of control cultures with the toxic agents TNFalpha, TNFalpha plus Actinomycin D, or Tat and gp120, induced neurotoxicity and death of neurons. Transduction of neurons with Ad. p35 blocked apoptosis, and eliminated cell death due to TNFalpha, or Tat and gp120. Viral vector transfer of the p35 gene efficiently protects human neurons from TNFalpha, or Tat and gp120-induced apoptosis and cell death. These results suggest that p35 transduction of neurons by viral vectors could be therapeutically useful in the treatment of human neurodegenerative diseases.     

Paxillin promotes breast tumor collective cell invasion through maintenance of adherens junction integrity

Distant organ metastasis is linked to poor prognosis during cancer progression. The expression level of the focal adhesion adapter protein paxillin varies among different human cancers, but its role in tumor progression is unclear. Herein we utilize a newly generated PyMT mammary tumor mouse model with conditional paxillin ablation in breast tumor epithelial cells, combined with in vitro three-dimensional (3D) tumor organoids invasion analysis and 2D calcium switch assays, to assess the roles for paxillin in breast tumor cell invasion. Paxillin had little effect on primary tumor initiation and growth but is critical for the formation of distant lung metastasis. In paxillin-depleted 3D tumor organoids, collective cell invasion was substantially perturbed. The 2D cell culture revealed paxillin-dependent stabilization of adherens junctions (AJ). Mechanistically, paxillin is required for AJ assembly through facilitating E-cadherin endocytosis and recycling and HDAC6-mediated microtubule acetylation. Furthermore, Rho GTPase activity analysis and rescue experiments with a RhoA activator or Rac1 inhibitor suggest paxillin is potentially regulating the E-cadherin-dependent junction integrity and contractility through control of the balance of RhoA and Rac1 activities. Together, these data highlight new roles for paxillin in the regulation of cell–cell adhesion and collective tumor cell migration to promote the formation of distance organ metastases.