HCCRBP-3 induces tumorigenesis through direct interaction with HCCR-1 in human cancers.

Human cervical cancer oncogene 1, HCCR-1, is over-expressed in various human tumors and appears to serve as a negative regulator of the p53 gene. HCCR-1 transgenic mice developed breast cancers but it is unknown how HCCR-1 contributes to tumorigenesis. We identified the HCCR-1 binding protein 3 (HCCRBP-3) as a binding partner for HCCR-1. These two proteins co-localized to the mitochondria. HCCRBP-3 over-expressed in various human tumors converted normal cells into tumor cells in vitro. Nude mice injected with NIH/3T3 cells stably transfected with HCCRBP-3 also induced the tumor formation. In addition, p53 showed the functional impairment in HCCRBP-3-transfected cells as accompanied with defective induction of p21 and bax. In support of this, p21 promoter activities containing p53 responsive elements were inhibited by HCCRBP-3 in a dose-dependent manner. Therefore, our study suggests that HCCRBP-3 contributes to the HCCR-1 induced tumorigenesis by interrupting the p53 function.

The functional and structural characterization of a novel oncogene GIG47 involved in the breast tumorigenesis.

BACKGROUND: A candidate oncogene GIG47, previously known as a neudesin with a neurotrophic activity, was identified by applying the differential expression analysis method. METHODS: As a first step to understand the molecular role of GIG47, we analyzed the expression profile of GIG47 in multiple human cancers including the breast cancer and characterized its function related to human carcinogenesis. Based on this oncogenic role of GIG47, we then embarked on determining the high-resolution structure of GIG47. We have applied multidimensional heteronuclear NMR methods to GIG47. RESULTS: GIG47 was over-expressed in primary breast tumors as well as other human tumors including carcinomas of the uterine cervix, malignant lymphoma, colon, lung, skin, and leukemia. To establish its role in the pathogenesis of breast cancer in humans, we generated stable transfectants of MCF7 cells. The ectopic expression of GIG47 in MCF7 cells promoted the invasiveness in the presence of 50% serum. In addition, it also resulted in the increased tumorigenicity in in vivo tumor formation assay. The tumorigenesis mechanism involving GIG47 might be mediated by the activation of MAPK and PI3K pathways. These results indicate that GIG47 plays a role in the breast tumorigenesis, thus representing a novel target for the treatment of breast cancer. To facilitate the development of GIG47-targeted therapeutics, we determined the structural configuration of GIG47. The high-resolution structure of GIG47 was obtained by combination of NMR and homology modeling. The overall structure of GIG47 has four α-helices and 6 ß-strands, arranged in a ß1-α1-ß2-ß3-α2-ß4-α3-α4-ß5-ß6 topology. There is a potential heme/steroid binding pocket formed between two helices α2 and α3. CONCLUSION: The determined three-dimensional structure of GIG47 may facilitate the development of potential anti-cancer agents.

Gremlin-1 induces BMP-independent tumor cell proliferation, migration, and invasion.

Gremlin-1, a bone morphogenetic protein (BMP) antagonist, is overexpressed in various cancerous tissues but its role in carcinogenesis has not been established. Here, we report that gremlin-1 binds various cancer cell lines and this interaction is inhibited by our newly developed gremlin-1 antibody, GRE1. Gremlin-1 binding to cancer cells was unaffected by the presence of BMP-2, BMP-4, and BMP-7. In addition, the binding was independent of vascular endothelial growth factor receptor-2 (VEGFR2) expression on the cell surface. Addition of gremlin-1 to A549 cells induced a fibroblast-like morphology and decreased E-cadherin expression. In a scratch wound healing assay, A549 cells incubated with gremlin-1 or transfected with gremlin-1 showed increased migration, which was inhibited in the presence of the GRE1 antibody. Gremlin-1 transfected A549 cells also exhibited increased invasiveness as well as an increased growth rate. These effects were also inhibited by the addition of the GRE1 antibody. In conclusion, this study demonstrates that gremlin-1 directly interacts with cancer cells in a BMP- and VEGFR2-independent manner and can induce cell migration, invasion, and proliferation.

Combined analysis of AFP and HCCR-1 as an useful serological marker for small hepatocellular carcinoma: a prospective cohort study.

Hepatocellular carcinoma (HCC) is one of the most frequent malignant tumors in the world. The only serological marker widely used for the diagnosis of HCC is alpha-fetoprotein (AFP). Despite that AFP is widely used for the diagnosis of HCC, it has a limit as a serological marker due to its low sensitivity and specificity. The human cervical cancer proto-oncogene 1 (HCCR-1) was previously reported as a new biomarker for HCC. To further evaluate the HCCR-1 as a biomarker for HCC, we conducted the prospective cohort study. We evaluated the significance of simultaneous measurement of 2 tumor markers in the diagnosis of HCC in China, Japan and Korea. Two markers for HCC, AFP and HCCR-1, were measured in the sera obtained from 1,338 patients at the time of initial diagnosis of HCC. Of the 1338 HCC patients, 616 (46%) and 686 (51.3%) were sero-positive for AFP and HCCR-1, respectively. The positive rate for HCC was increased up to 74.1% in combined use of AFP and HCCR-1. Many cases (54%) for AFP-negative HCC were positive for HCCR-1 and vice versa. More importantly, the diagnostic rate for small HCC (< 2 cm) was significantly improved in the combined analysis of AFP and HCCR-1 to 56.9% although it was only 40.1% and 23.4% in the single analysis of HCCR-1 and AFP, respectively. Our result suggests that the HCCR-1 could be an useful biomarker for HCC while the diagnostic rate could be significantly improved in the combined use of HCCR-1 and AFP.

Simultaneous measurements of serum AFP, GPC-3 and HCCR for diagnosing hepatocellular carcinoma.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is a prevalent malignant tumor. Tumor markers are very useful in early diagnosis; however a single marker is rather limited. We launched a test to increase the diagnostic sensitivity through the combined detection. METHODOLOGY: Serum concentration of three tumor-markers, Glypican-3 (GPC-3), Human-Cervical-Cancer-Oncogene (HCCR) and a-fetoprotein (AFP), were determined in 189 samples: 101 cases of HCC, 40 cases of cirrhosis, 18 cases of hepatitis and 30 cases of control healthy donors. Every marker was evaluated for its diagnostic value by one-way-analysis-of-variance and receiver-operating-characteristics analysis. RESULTS: GPC-3 was the best marker with an area under the curve (AUC) of 0.892; using 26.8ng/mL as the cut-off for HCC diagnosis, GPC-3 has a sensitivity of 51.5% and maintains a specificity of 92.8%. HCCR, with an AUC of 0.831, can reach a sensitivity of 22.8% and maintain a specificity of 90.9% if the cut-off is set as 58.8mAU/mL. With an AUC of 0.827, the efficacy and sensitivity of AFP were 36.6% and 98.5% when using 199.3ng/mL as the cut-off. No significant correlation was found between these three markers. Simultaneously detecting three markers can significantly increases the sensitivity to 80.2%, much higher than AFP alone. CONCLUSIONS: GPC-3 and HCCR are useful tumor markers complementary to AFP for clinical diagnosis of HCC.

Clinical utility of alpha fetoprotein and HCCR-1, alone or in combination, in patients with chronic hepatitis, liver cirrhosis and hepatocellular carcinoma.

Serum alpha fetoprotein (AFP) is the most widely used tumor marker in detecting patients with hepatocellular carcinoma (HCC). However, it has been indicated that HCCR-1 (human cervical cancer oncogene 1) might be supplementary to AFP in the detection. We conducted a prospective study in 120 normal and 524 liver disease patients to evaluate the significance of simultaneous measurement of 2 tumor markers (AFP and HCCR-1) in the diagnosis of HCC through the cohort study in Korea and China. We also performed immunohistochemical studies using 25 normal subjects (N), 32 liver cirrhosis (LC) and 116 HCC tissues. The sensitivities of AFP (20 ng/mL) and HCCR-1 (10 ng/mL) in HCC were 55.8% (164/294) and 44.2% (130/294), respectively. When AFP was combined with HCCR-1, sensitivities increased to 4.2% (N), 12.7% (chronic hepatitis; CH), 50.0% (LC), and 77.2% (HCC), respectively. Although there was no significant difference in the diagnostic rate for HCC between AFP and HCCR-1, many cases for AFP-negative HCC were positive for HCCR-1 and vice versa. Moreover, the combined use of AFP and HCCR-1 improved the diagnostic rate to 70.8% in small HCC (< 2 cm) and 81.6% in large HCC (⩾ 2 cm), respectively. AFP and HCCR-1 are independent markers. Our result suggests that the HCCR-1 could be an useful biomarker for HCC while the diagnostic rate could be significantly improved in the combined use of HCCR-1 and AFP.

The prognostic potential of keratin 18 in breast cancer associated with tumor dedifferentiation, and the loss of estrogen and progesterone receptors.

The differential expression profiling with breast normal and tumor tissues, and a breast cancer cell line led to identification of cytokeratin 18 (KT18) gene over-expressed in breast cancer. The expression pattern of KT18 in breast cancer was compared to those of conventional tumor markers such as proliferating cell nuclear antigen (PCNA) and minichromosome maintenance protein 3 (MCM3). Their expression patterns in breast cancer were almost identical, suggesting that KT18 might be useful for detection of proliferating fractions in the breast cancer. The immunohistochemical analyses on the tissue microarray consisting of invasive ductal carcinomas revealed that the up-regulation of KT18 is observed in a majority of breast carcinomas whereas its down-regulation also occurs at a less frequency. Of particular interest, KT18 down-regulation was associated with histologically poorly differentiated carcinomas than well differentiated carcinomas. In addition, it was also significantly associated with the loss of estrogen receptor (ER) and progesterone receptor (PR), the prognostic markers of the breast cancer (P < 0.05), while not with HER2, tumor size, and lymph node metastasis. This result suggests that KT18 correlated with ER and PR may be utilized for the prognosis of breast cancer. Furthermore, the forced down-regulation of KT18 enhanced the growth of tumor xenografts in vivo and invasiveness in vitro. Therefore, our findings suggest that loss of KT18 expression might be a good indicator of the poor prognosis of the breast cancer and it may play an active role in the breast tumorigenesis.

Transdifferentiation-inducing HCCR-1 oncogene.

BACKGROUND: Cell transdifferentiation is characterized by loss of some phenotypes along with acquisition of new phenotypes in differentiated cells. The differentiated state of a given cell is not irreversible. It depends on the up- and downregulation exerted by specific molecules. RESULTS: We report here that HCCR-1, previously shown to play an oncogenic role in human cancers, induces epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) in human and mouse, respectively. The stem cell factor receptor CD117/c-Kit was induced in this transdifferentiated (EMT) sarcoma tissues. This MET occurring in HCCR-1 transfected cells is reminiscent of the transdifferentiation process during nephrogenesis. Indeed, expression of HCCR-1 was observed during the embryonic development of the kidney. This suggests that HCCR-1 might be involved in the transdifferentiation process of cancer stem cell. CONCLUSIONS: Therefore, we propose that HCCR-1 may be a regulatory factor that stimulates morphogenesis of epithelia or mesenchyme during neoplastic transformation.

p-Glycoprotein ABCB5 and YB-1 expression plays a role in increased heterogeneity of breast cancer cells: correlations with cell fusion and doxorubicin resistance.

BACKGROUND: Cancer cells recurrently develop into acquired resistance to the administered drugs. The iatrogenic mechanisms of induced chemotherapy-resistance remain elusive and the degree of drug resistance did not exclusively correlate with reductions of drug accumulation, suggesting that drug resistance may involve additional mechanisms. Our aim is to define the potential targets, that makes drug-sensitive MCF-7 breast cancer cells turn to drug-resistant, for the anti-cancer drug development against drug resistant breast cancer cells. METHODS: Doxorubicin resistant human breast MCF-7 clones were generated. The doxorubicin-induced cell fusion events were examined. Heterokaryons were identified and sorted by FACS. In the development of doxorubicin resistance, cell-fusion associated genes, from the previous results of microarray, were verified using dot blot array and quantitative RT-PCR. The doxorubicin-induced expression patterns of pro-survival and pro-apoptotic genes were validated. RESULTS: YB-1 and ABCB5 were up regulated in the doxorubicin treated MCF-7 cells that resulted in certain degree of genomic instability that accompanied by the drug resistance phenotype. Cell fusion increased diversity within the cell population and doxorubicin resistant MCF-7 cells emerged probably through clonal selection. Most of the drug resistant hybrid cells were anchorage independent. But some of the anchorage dependent MCF-7 cells exhibited several unique morphological appearances suggesting minor population of the fused cells maybe de-differentiated and have progenitor cell like characteristics. CONCLUSION: Our work provides valuable insight into the drug induced cell fusion event and outcome, and suggests YB-1, GST, ABCB5 and ERK3 could be potential targets for the anti-cancer drug development against drug resistant breast cancer cells. Especially, the ERK-3 serine/threonine kinase is specifically up-regulated in the resistant cells and known to be susceptible to synthetic antagonists.

Minichromosome maintenance protein 3 is a candidate proliferation marker in papillary thyroid carcinoma.

The proliferative capacity of tumor cells is a characteristic feature in the whole growing tumors. Many pathologists and clinicians have used the estimation of cell proliferation for prognostic information. Minichromosome maintenance protein 3 (MCM3) is known to have a role on the initiation and regulation of DNA replication during cell cycle. The aim of this study was to evaluate the potential applicability of one of the MCM proteins, MCM3, as a proliferation marker in papillary thyroid carcinoma (PTC) with correlation to clinicopathological parameters. We performed the immunohistochemical analysis for MCM3 and Ki-67 in 60 cases of PTC and Western blot analysis for MCM3 expression in 6 PTCs and normal thyroid tissues. The comparison of MCM3 labeling index (LI) to tumor size (P=0.031) and extrathyroidal extension (P=0.037) was statistically significant while that of Ki-67 LI to them was not. Moreover, a significant association was not observed between MCM3 and Ki-67, but the MCM3 LI was considerably higher. Western blot analyses revealed that the MCM3 protein expression levels were overexpressed in all PTCs. On the contrary, the levels of MCM3 were very low or absent in all normal thyroid tissues. Our results indicate that MCM3 may be a more reliable proliferation marker than Ki-67 in accessing the growth of tumor and evaluating tumor aggressiveness of PTC.

TCF/beta-catenin plays an important role in HCCR-1 oncogene expression.

BACKGROUND: Oncogene HCCR-1 functions as a negative regulator of the p53 and contributes to tumorigenesis of various human tissues. However, it is unknown how HCCR-1 contributes to the cellular and biochemical mechanisms of human tumorigenesis. RESULTS: In this study, we showed how the expression of HCCR-1 is modulated. The luciferase activity assay indicated that the HCCR-1 5'-flanking region at positions -166 to +30 plays an important role in HCCR-1 promoter activity. Computational analysis of this region identified two consensus sequences for the T-cell factor (TCF) located at -26 to -4 (Tcf1) and -136 to -114 (Tcf2). Mutation at the Tcf1 site led to a dramatic decrease in promoter activity. Mobility shift assays (EMSA) revealed that nuclear proteins bind to the Tcf1 site, but not to the Tcf2 site. LiCl, Wnt signal activator by GSK-3beta inhibition, significantly increased reporter activities in wild-type Tcf1-containing constructs, but were without effect in mutant Tcf1-containing constructs in HEK/293 cells. In addition, endogenous HCCR-1 expression was also increased by treatment with GSK-3beta inhibitor, LiCl or AR-A014418 in HEK/293 and K562 cells. Finally, we also observed that the transcription factor, TCF, and its cofactor, beta-catenin, bound to the Tcf1 site. CONCLUSION: These findings suggest that the Tcf1 site on the HCCR-1 promoter is a major element regulating HCCR-1 expression and abnormal stimulation of this site may induce various human cancers.

Oncoprotein HCCR-1 expression in breast cancer is well correlated with known breast cancer prognostic factors including the HER2 overexpression, p53 mutation, and ER/PR status.

BACKGROUND: Oncoprotein HCCR-1 functions as a negative regulator of the p53 and contributes breast tumorigenesis. The serum HCCR-1 assay is useful in diagnosing breast cancer and mice transgenic for HCCR developed breast cancers. But it is unknown how HCCR-1 contributes to human breast tumorigenesis. METHODS: Oncogene HCCR-1 expression levels were determined in normal breast tissues, breast cancer tissues and cancer cell lines. We examined whether HCCR-1 protein expression in breast cancer is related to different biological characteristics, including ER, PR, p53 genotype, and HER2 status in 104 primary breast cancer tissues using immunohistochemical analyses. RESULTS: HCCR-1 was upregulated in breast cancer cells and tissues compared with normal breast tissues. In this study, overexpression of HCCR-1 was well correlated with known breast cancer prognostic markers including the presence of steroid receptors (ER and PR), p53 mutation and high HER2 overexpression. HCCR-1 was not detected in the ER-negative, PR-negative, p53 negative and low HER2 breast cancer tissues. These data indicate that the level of HCCR-1 in breast cancer tissues is relatively well correlated with known breast cancer factors, including the HER2 overexpression, p53 mutation, and ER/PR status. CONCLUSION: Determination of HCCR-1 levels as options for HER2 testing is promising although it needs further evaluation.

Dual action of apolipoprotein E-interacting HCCR-1 oncoprotein and its implication for breast cancer and obesity.

Obese women have an increased risk for post-menopausal breast cancer. The physiological mechanism by which obesity contributes to breast tumourigenesis is not understood. We previously showed that HCCR-1 oncogene contributes to breast tumourigenesis as a negative regulator of p53 and detection of HCCR-1 serological level was useful for the diagnosis of breast cancer(.) In this study, we found that the HCCR-1 level is elevated in breast cancer tissues and cell lines compared to normal breast tissues. We identified apolipoprotein E (ApoE) interacting with HCCR-1. Our data show that HCCR-1 inhibits anti-proliferative effect of ApoE, which was mediated by diminishing ApoE secretion of breast cancer cells. Finally, HCCR-1 induced the severe obesity in transgenic mice. Those obese mice showed severe hyperlipidaemia. In conclusion, our results suggest that HCCR-1 might play a role in the breast tumourigenesis while the overexpression of HCCR-1 induces the obesity probably by inhibiting the cholesterol-lowering effect of ApoE. Therefore, HCCR-1 seems to provide the molecular link between the obesity and the breast cancer risk.

Both plasma retinol-binding protein and haptoglobin precursor allele 1 in CSF: candidate biomarkers for the progression of normal to mild cognitive impairment to Alzheimer's disease.

Cerebrospinal fluid (CSF) may be of valuable for exploring protein markers for the diagnosis of Alzheimer's disease (AD). The prospect of early detection and treatment, to slow progression, holds hope for aging populations with increased average lifespan. The aim of the present study was to investigate candidate CSF biological markers in patients with mild cognitive impairment (MCI) and AD and compare them with age-matched normal control subjects. In this report, we applied proteomics approaches to analyze 60 CSF samples derived from patients with neurodegenerative diseases such as MCI and AD. We classified patients by three groups: normal controls without cognitive dysfunction, MCI and AD. The AD group was subdivided into three groups by clinical severity according to clinical dementia rating (CDR), a well known clinical scale for dementia. We demonstrated a gradual decrease or absent of plasma retinol-binding protein (RBP) and haptoglobin precursor allele 1 in CSF from patients with MCI and AD compared to the age-matched normal subjects. Moreover, expression levels of both RBP and haptoglobin precursor allele 1 were observed to be very high in age-matched normal subjects. In contrast, the RBP and haptoglobin precursor allele 1 were much decreased in the MCI group; those expressions were more weak or absent in AD group, and correlated with disease severity and progression. These findings suggest that the CSF levels of both RBP and haptoglobin precursor allele 1 may be candidate biomarkers for the progression of normal to MCI to AD.

HCCRBP-1 directly interacting with HCCR-1 induces tumorigenesis through P53 stabilization.

Oncogene HCCR-1 functions as a negative regulator of the p53 and contributes to tumorigenesis of various human tissues. HCCR transgenic mice developed breast cancers but it is unknown how HCCR-1 contributes to human tumorigenesis. This study identified a HCCR-1-binding protein 1 (HCCRBP-1) as an HCCR binding partner by performing yeast two hybrid screening. Their endogenous interaction was further confirmed by coimmunoprecipitation experiments. These two proteins colocalized in the mitochondria. HCCRBP-1 was overexpressed in various human tumors. In addition, HCCRBP-1 alone converted NIH/3T3 cells into tumor cells in combination with no other oncogenes. HCCRBP-1 induced tumorigenesis by markedly activating PKC activities but decreasing the pro-apoptotic PKC alpha and PKC delta isoform levels. We observed that p53 stabilization also occurred with functional impairment in HCCRBP-1-transfected 293 cells, as indicated by defective induction of p21, MDM2 and bax. Indeed, HCCRBP-1 decreased p21 promoter activity probably via p53 stabilization leading to the defective function. These results indicate that HCCRBP-1 oncogene induces p53 stabilization and thereby contributes to tumorigenesis.

HCCR-1, a novel oncogene, encodes a mitochondrial outer membrane protein and suppresses the UVC-induced apoptosis.

BACKGROUND: The Human cervical cancer oncogene (HCCR-1) has been isolated as a human oncoprotein, and has shown strong tumorigenic features. Its potential role in tumorigenesis may result from a negative regulation of the p53 tumor suppressor gene. RESULTS: To investigate the biological function of HCCR-1 in the cell, we predicted biological features using bioinformatic tools, and have identified a LETM1 homologous domain at position 75 to 346 of HCCR-1. This domain contains proteins identified from diverse species predicted to be mitochondrial proteins. Fluorescence microscopy and fractionation experiments showed that HCCR-1 is located in mitochondria in the COS-7, MCF-7 and HEK/293 cell lines, and subcompartamentally at the outer membrane in the HEK/293 cell line. The topological structure was revealed as the NH2-terminus of HCCR-1 oriented toward the cytoplasm. We also observed that the D1-2 region, at position 1 to 110 of HCCR-1, was required and sufficient for posttranslational mitochondrial import. The function of HCCR-1 on mitochondrial membrane is to retard the intrinsic apoptosis induced by UVC and staurosporine, respectively. CONCLUSION: Our experiments show the biological features of HCCR-1 in the cell, and suggest that uncontrolled expression of HCCR-1 may cause mitochondrial dysfunction that can result in resisting the UVC or staurosporine-induced apoptosis and progressing in the tumor formation.

Fibrinogen gamma-A chain precursor in CSF: a candidate biomarker for Alzheimer's disease.

BACKGROUND: Cerebrospinal fluid (CSF) may be valuable for exploring protein markers for the diagnosis of Alzheimer's disease (AD). The prospect of early detection and treatment, to slow progression, holds hope for aging populations with increased average lifespan. The aim of the present study was to investigate candidate CSF biological markers in patients with mild cognitive impairment (MCI) and AD and compare them with age-matched normal control subjects. METHODS: We applied proteomics approaches to analyze CSF samples derived from 27 patients with AD, 3 subjects with MCI and 30 controls. The AD group was subdivided into three groups by clinical severity according to clinical dementia rating (CDR), a well known clinical scale for dementia. RESULTS: We demonstrated an elevated level of fibrinogen gamma-A chain precursor protein in CSF from patients with mild cognitive impairment and AD compared to the age-matched normal subjects. Moreover, its expression was more prominent in the AD group than in the MCI and correlated with disease severity and progression. In contrast, fibrinogen gamma-A chain precursor protein was detected very low in the age-matched normal group. CONCLUSION: These findings suggest that the CSF level of fibrinogen gamma-A chain precursor may be a candidate biomarker for AD.

Intestinal IgA synthesis: a primitive form of adaptive immunity that regulates microbial communities in the gut.

Our intestine is colonized by an impressive community of bacteria, that has profound effects on the immune functions. The relationship between gut microbiota and the immune system is one of reciprocity: bacteria have important contribution in nutrient processing and education of the immune system and conversely, the immune system, particularly gut-associated lymphoid tissues (GALT) plays a key role in shaping the repertoire of gut microbiota. In this review we discuss new insights into the role of IgA in the maintenance of immune homeostasis and the reciprocal interactions between gut B cells and intestinal bacteria.

Regulation of B1 cell migration by signals through Toll-like receptors.

Peritoneal B1 cells are known to generate large amounts of antibodies outside their residential site. These antibodies play an important role in the early defense against bacteria and viruses, before the establishment of adaptive immune responses. Although many stimuli, including antigen, lipopolysaccharide, or cytokines, have been shown to activate B1 cells and induce their differentiation into plasma cells, the molecular signals required for their egress from the peritoneal cavity are not understood. We demonstrate here that direct signals through Toll-like receptors (TLRs) induce specific, rapid, and transient down-regulation of integrins and CD9 on B1 cells, which is required for detachment from local matrix and a high velocity movement of cells in response to chemokines. Thus, we revealed an unexpected role for TLRs in governing the interplay between integrins, tetraspanins, and chemokine receptors required for B1 cell egress and, as such, in facilitating appropriate transition from innate to adaptive immune responses.