PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of jbcThe Journal of Biological Chemistry
 
J Biol Chem. 2012 February 17; 287(8): 5483–5491.
Published online 2011 December 27. doi:  10.1074/jbc.M111.324632
PMCID: PMC3285325

This article has been retractedRetraction in: J Biol Chem. 2013 September 6; 288(36): 26177    See also: PMC Retraction Policy

Mechanism of MTA1 Protein Overexpression-linked Invasion

MTA1 REGULATION OF HYALURONAN-MEDIATED MOTILITY RECEPTOR (HMMR) EXPRESSION AND FUNCTION*An external file that holds a picture, illustration, etc.
Object name is sbox.jpg

Abstract

Even though the hyaluronan-mediated motility receptor (HMMR), a cell surface oncogenic protein, is widely up-regulated in human cancers and correlates well with cell motility and invasion, the underlying molecular and nature of its putative upstream regulation remain unknown. Here, we found for the first time that MTA1 (metastatic tumor antigen 1), a master chromatin modifier, regulates the expression of HMMR and, consequently, its function in breast cancer cell motility and invasiveness. We recognized a positive correlation between the levels of MTA1 and HMMR in human cancer. Furthermore, MTA1 is required for optimal expression of HMMR. The underlying mechanism includes interaction of the MTA1·RNA polymerase II·c-Jun coactivator complex with the HMMR promoter to stimulates its transcription. Accordingly, selective siRNA-mediated knockdown of HMMR in breast cancer cells substantially reduces the invasion and migration of cells. These findings reveal a regulatory role for MTA1 as an upstream coactivator of HMMR expression and resulting biological phenotypes.

Keywords: Chromatin Regulation, Chromatin Remodeling, Coactivator Transcription, Gene Regulation, Invasion, Migration, Breast Cancer, HMMR, MTA1

Introduction

Cancer progression is a complex process consisting of transformation, tumor growth, invasion, and metastasis. Cancer invasion and metastasis, both integral components of cancer progression, require an active motile cell phenotype. Cell motility and invasion are tightly regulated by the growth factors, cytokines, and extracellular matrix components in the tumor microenvironment (1, 2). Hyaluronic acid (HA),4 a component of the extracellular matrix, is abundantly secreted from the stromal fibroblast of cancerous tissues in response to humoral factors derived from tumor cells (3). HA also participates in the stimulation of breast cancer progression and invasion (48). The cell surface hyaluronan-mediated motility receptor (HMMR; CD168) and CD44 are representative HA receptors (9). Furthermore, the net expression of HMMR is up-regulated in advanced cancers, including breast (10), endometrial (11), colon (12), oral squamous cell carcinoma and multiple myeloma (13), and prostate (14), and its expression is generally believed to be essential for cancer progression (9, 15). In cancer cells, HMMR is stimulated upon engagement of HA-driven intracellular signaling and stimulation of microfilament formation that promotes cell motility (1619). Extracellular HMMR activates ERK1/2 signaling through phosphorylation of the Src and focal adhesion kinases with the association of CD44 or the PDGF receptor (18). HMMR also induces invasion through myosin light chain phosphorylation by increasing the GTPase activity (19). HMMR is distributed in both the cytoplasm and nucleus and functions as a cell surface receptor for HA. In addition, HMMR is a centrosomal protein that contributes to the stability of the mitotic spindle (16, 17). In addition, HMMR also regulates the G2/M phase in the cell cycle (16). These findings highlight the significance of HMMR in cancer invasion and metastasis.

Chromatin remodeling is a dynamic process that tightly regulates gene transcription and expression, consequently resulting in phenotypic changes. The process of chromatin remodeling requires activation of transcription factors and coregulators (20). MTA1 (metastatic tumor antigen 1), a founding member of the MTA family of chromatin modifiers, forms an integral part of the NuRD (nucleosome remodeling and histone deacylation) complex, which is involved in transcriptional regulation, histone deacetylation, and chromatin remodeling. MTA1 executes its function on the target gene by recruiting either RNA polymerase II (pol II) or histone deacetylase to the target gene chromatin (2124). MTA1 is up-regulated in human cancers and also acts as an oncogene (2527). MTA1 forms a coactivation complex with c-Jun and pol II on FosB motif-containing chromatin and plays a pivotal role in invasion by repressing E-cadherin expression (28). MTA1 also increases the transcriptional activity of hypoxia-inducible factor-1α and the expression of VEGF-A, which contribute to the process of metastasis (29, 30). A recent report also suggests the involvement of MTA1 in invasion of colorectal cancer cells by increasing the expression of VEGF-C (31). Although there is overexpression of HMMR and MTA1 during cancer invasion and metastasis, the existence of a relationship between these molecules remains unclear.

Recently, this laboratory showed that depletion of MTA1 results in decreased expression of HMMR mRNA (32). Because of the established role of HMMR in cancer invasion and metastasis, we made an attempt here to delineate the role of MTA1 in the regulation of HMMR expression and function. We discovered that MTA1 is an upstream coactivator of HMMR and is needed for its expression and resulting functions in invasion and migration.

EXPERIMENTAL PROCEDURES

Cell Culture and Antibodies

All of the cell lines used in this study were from the laboratory of R. K. and have been used previously (22, 33, 34). All of the cells were cultured in DMEM supplemented with 10% FBS and 1× antibiotic solution. Antibodies against MTA1 (A300-280A-1) and pol II (A300-653A) were purchased from Bethyl Laboratories (Montgomery, TX), and anti-CD168/HMMR (ab87677) antibody was purchased from Abcam (Cambridge, MA). Normal mouse IgG, rabbit IgG, and antibodies against actin and vinculin were obtained from Sigma, and anti-c-Jun antibody was purchased form Santa Cruz Biotechnology (Santa Cruz, CA).

Microarray Analysis

A microarray data set (accession number GSE26304) was obtained from the NCBI Gene Express Omnibus (GEO) Database consisting of breast cancer samples from 31 pure ductal carcinoma in situ (DCIS) patients, 36 invasive ductal carcinoma patients, and 42 mixed DCIS patients. The Agilent two-color raw files obtained from the GEO Database were imported into the GeneSpring GX 11.0 software package (Agilent Technologies) for quality control and statistical analysis of the data. The raw expression values were normalized using the robust multichip average algorithm. Two probes, A_23_P9513 and A_24_P241370, were used to detect the levels of MTA1, and one probe, A_23_P70007, was used to detect the levels of HMMR. The robust multichip average-derived log-transformed expression values for each probe were then averaged, and the mean values were used for further analysis. The single-factor analysis of variance test was used to compare the relative transcript levels among the different groups, and Pearson's correlation coefficient was used to determine the correlation between the MTA1 and HMMR transcript levels.

Western Blot Analysis, Quantitative RT-PCR (qPCR), and Reporter Assays

Western blot analysis, qPCR, and reporter assays were carried out as described elsewhere (24). The primers used for qPCR analysis are provided in supplemental Table 1.

siRNA Transfection

siRNAs against MTA1 (M-004127-01), HMMR (M-010409-01-005), and non-targeting control siRNA (D-001206-05) were purchased from Dharmacon RNAi Technology (Lafayette, CO). A second siRNA against MTA1 (sc-35981) was obtained from Santa Cruz Biotechnology. Cells were seeded in a 6-well plate at 40% confluency on the day before transfection. Transfections were performed according to the manufacturer's protocol using Oligofectamine (Invitrogen) with a final concentration of 200 nm siRNA. Transfected cells were collected after 48 h, and cell lysates were prepared as described previously (28).

Chromatin Immunoprecipitation Assay

ChIP assay was performed according to the protocol described previously (28). Briefly, cells were cross-linked with formaldehyde (1% final concentration) and sonicated on ice to fragment the chromatin into an average length of 500 bp to 1 kb. The lysates were diluted using chromatin dilution buffer. Anti-MTA1, anti-c-Jun, or mouse IgG antibodies were used to immunoprecipitate the respective antigens at 4 °C overnight. Protein A-Sepharose beads saturated with bovine serum albumin and single-stranded DNA were added to the lysate to isolate the antibody-bound complexes. The beads were washed to remove nonspecific binding, and the antibody-bound chromatin was eluted. The eluate was “de-cross-linked” by heating at 65 °C for 6 h. RNase was added during this step to digest the RNA contaminants. Samples were treated with proteinase K for 1 h at 45 °C to digest the proteins pulled down by immunoprecipitation, and finally, the DNA was extracted using the phenol/chloroform method. For the double-ChIP experiment, an initial ChIP assay was done with anti-MTA1 antibody to immunoprecipitate MTA1-bound chromatin, which was eluted from the protein A-Sepharose beads and subjected to a second ChIP assay with either anti-pol II or anti-c-Jun antibody. With the DNA eluted at the end of the ChIP analysis, PCR and qPCR were performed using the primers listed in supplemental Table 2.

Electrophoretic Mobility Shift Assay

Nuclear extracts were prepared using a Nonidet P-40 lysis method. EMSA for HMMR promoter binding was performed using the annealed and [γ-32P]ATP end-labeled oligonucleotides in a 20-μl reaction mixture for 15 min at 20 °C. Samples were run on a nondenaturing 5% polyacrylamide gel and imaged by autoradiography. Specific competitions were performed by adding a 100-molar excess of competitor to the incubation mixture, and supershift EMSAs were performed by adding the indicated antibodies. The oligonucleotides used were listed in supplemental Table 3.

Confocal Microscopy

The surface accumulation of proteins was determined by immunofluorescence. MCF-7 and MDA-MB-231 cells were grown on sterile coverslips and transfected with MTA1 siRNA using Oligofectamine following the manufacturer's protocol. The MTA1 siRNA-transfected cells were fixed using 4% paraformaldehyde for 10 min, permeabilized using 0.1% Triton-X 100 for 7 min, and blocked with 10% normal goat serum in PBS. The cells were incubated overnight with mouse anti-MTA1 monoclonal antibody (sc-17773, Santa Cruz Biotechnology) and rabbit anti-CD168 polyclonal antibody (ab87677) at 4 °C, washed twice with PBS, and incubated with anti-mouse and anti-rabbit secondary antibodies conjugated with Alexa Fluor 488 and Alexa Fluor 555, respectively. DAPI (Molecular Probes, Eugene, OR) was used as for nuclear staining. The images were obtained with an Olympus FV300 laser-scanning confocal microscope using sequential laser excitation to minimize fluorescence emission bleed-through.

Boyden Chamber Assay and Wound Healing Assay

Migration and invasion assays were performed as described previously (35). In brief, MCF-7 cells were transfected with HMMR siRNA, and after 48 h of transfection, cells were trypsinized, washed with PBS, and resuspended in DMEM in the presence of 0.1% bovine serum albumin. For the invasion assay, cells were loaded into the upper well of an uncoated Boyden chamber (BD Biosciences) at a concentration of 1 × 105 cells/well. The lower side of the separating filter was filled with conditioned medium from NIH3T3 cells. The invasion assay was performed as described for the migration assay except that we used Matrigel-coated Boyden chambers (BD Biosciences). Cells were stained with DAPI and imaged with an Olympus IX71 inverted microscope using DP2-BSW application software (Olympus Imaging America Inc., Center Valley, PA). Cell numbers for migration and invasion were then determined by counting the number of cells present in 15 microscope fields at ×20 magnification per insert. For the wound healing assay, each plate received multiple “wounds” with a 200-μl pipette tip. After an additional 24 h, each plate was examined by phase-contrast microscopy for the amount of wound closure by measuring the physical separation remaining between the original wound widths using the Olympus DP2-BSW digital camera software. Ten separate measurements were made per plate, and each experiment was performed in triplicate.

RESULTS AND DISCUSSION

MTA1 Expression Correlates with Levels of HMMR

A recent gene profiling study of wild-type murine embryonic fibroblasts (MEFs) and those depleted of the mta1 gene (mta1−/− MEFs) revealed the existence of a number of new MTA1-regulated genes (32). One of the targets of interest for MTA1 was HMMR. Because both MTA1 (2527) and HMMR (1014, 16, 36, 37) are widely up-regulated genes in human cancers, we undertook this study to understand the biochemical basis of the noted MTA1-HMMR association. Western blot analysis of breast cancer cell lines ZR-75, MCF-7, and T47D revealed a positive correlation between the levels of MTA1 and HMMR proteins (Fig. 1A). Higher levels of MTA1 and HMMR were observed in the T47D cells, whereas the levels were modest in ZR-75 and MCF-7 cells (Fig. 1A). Furthermore, the level of MTA1 correlated well with the status of HMMR in the triple-negative breast cancer cell lines (Fig. 1B). In addition, meta-analysis of microarray data sets from the GEO Database revealed a strong correlation between the levels of MTA1 and HMMR in breast tumors (Fig. 1, C–E). A microarray data set (accession number GSE26304) consisting of breast cancer samples from 31 pure DCIS patients, 36 invasive ductal carcinoma patients, and 42 mixed DCIS patients was obtained from the NCBI GEO Database (www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE26304) to determine the correlation between MTA1 and HMMR. Two probes, A_23_P9513 and A_24_P241370, were used to detect the levels of MTA1, and one probe, A_23_P70007, was used to detect the levels of HMMR. Analysis of the z-score normalization for HMMR and MTA1 (Fig. 1, C and D) revealed a positive correlation between the levels of MTA1 and HMMR mRNAs (Fig. 1E).

FIGURE 1.
MTA1 expression correlates with HMMR expression in breast cancer progression. A, left panel, Western blot analysis of MTA1 and HMMR protein expression from cell lysates of the ZR-75, MCF-7, and T47D breast cancer cell lines. Right panel, densitometric ...

We found that the noted correlation between the levels of MTA1 and HMMR was not limited to breast cancer, as we also noticed a strong correlation between the levels of MTA1 and HMMR mRNAs and proteins in prostate cancer cell lines (Fig. 2A). Meta-analysis of the prostate microarray data set (GSE6919) indicated a definitive correlation between the levels of MTA1 and HMMR. The data set included 18 normal prostate tissue samples, 63 normal prostate tissues adjacent to tumors, 65 primary prostate tumor samples, and 25 metastatic prostate tumor samples. Three probes (1642_at, 1643_g_at, and 38633_at) for MTA1 and one probe (36863_at) for HMMR was used. Using the z-score normalization procedure, the transcript levels of MTA1 and HMMR were normalized (Fig. 2, B and C), and Pearson analysis revealed a positive relationship between the levels of MTA1 and HMMR (Fig. 2D).

FIGURE 2.
MTA1 expression correlates with HMMR expression in prostate cancer progression. A, qPCR and Western blot analyses of the expression of MTA1 and HMMR in the invasive prostate cancer cell lines LNCap, VCap, and MDA-PCb-2a. The mRNA expression levels of ...

MTA1 Regulates HMMR Expression

Because of the existence of a strong correlation between the expression levels of MTA1 and HMMR, we investigated the contribution of MTA1 to the regulation of HMMR. First, we evaluated the effect of MTA1 depletion on the expression levels of HMMR in both the MEFs and HeLa cells. Because of low expression levels of HMMR in normal cells (9, 15, 38, 39), we were unable to observe HMMR protein in MEFs (Fig. 3A). However, siRNA-mediated knockdown of MTA1 in the HeLa cells effectively decreased the levels of HMMR (Fig. 3A). In addition, we observed an increased expression of HMMR in the MCF-7 cells stably overexpressing MTA1 (Fig. 3B). Consistent with these results, we found decreased levels of HMMR mRNA after MTA1 knockdown in the HeLa cells (Fig. 3, C and D) and an increased level of HMMR mRNA in the MCF-7 cells stably expressing MTA1 (Fig. 3E). As MTA1 positively regulates the expression of HMMR, we next analyzed the levels of MTA1 and HMMR in a widely studied experimental model of breast cancer progression involving isogenic MCF-10A cells (non-malignant), MCF-10AT cells (weakly tumorigenic), MCF-10CA1D cells (undifferentiated metastatic), and MCF-10DCIS cells (highly proliferative, aggressive, and invasive) (35). The levels of both the MTA1 and HMMR proteins and mRNAs were progressively up-regulated from noninvasive MCF-10A cells to highly invasive MCF-10DCIS cells (Fig. 3F). Taken together, these results suggest that MTA1 regulates the expression of HMMR.

FIGURE 3.
MTA1 regulates HMMR expression. A, Western blot analysis of MTA1 and HMMR protein expression in mta1+/+ and mta1−/− MEFs and in HeLa cells after MTA1 knockdown using MTA1-specific siRNAs 1 and 2 independently. MTA1 siRNAs 1 and 2 were ...

To understand the basis of MTA1 regulation of HMMR, we next investigated the HMMR promoter activity. We observed decreased HMMR transcription in the mta1−/− MEFs, whereas this effect could be effectively rescued by reintroduction of MTA1 (Fig. 4A). Consistent with these observations, siRNA-mediated knockdown of MTA1 in the MCF-7 cells also down-regulated HMMR transcription (Fig. 4B), whereas MTA1 overexpression in the MCF-7 cells stimulated the HMMR promoter activity (Fig. 4C). These findings suggest a role for MTA1 in the regulation of HMMR transcription.

FIGURE 4.
MTA1 regulates HMMR transcription. A, HMMR promoter-luciferase (HMMR Pro-luc) activity in wild-type and mta1−/− MEFs and in mta1−/− MEFs overexpressing the mta1 gene. These cells were transfected with the human HMMR-pGL4.1 ...

MTA1·pol II·c-Jun Coactivator Complex Activates HMMR Transcription

To gain a deeper insight into the molecular mechanism underlying MTA1 regulation of HMMR expression, we carried out a detailed ChIP analysis in the MCF-7 cells and mapped the recruitment of MTA1 to six regions of the HMMR promoter (Fig. 5A). We identified the recruitment of MTA1 to only three regions of the HMMR promoter (Fig. 5B). Because MTA1 increases the transcription of HMMR, we analyzed the association of MTA1 with pol II by double-ChIP assay. Interestingly, we observed the recruitment of MTA1 and the pol II coactivator complex to only region I (−321 to −102) of the HMMR promoter (Fig. 5C).

FIGURE 5.
Recruitment of MTA1·pol II·c-Jun coactivator complex to HMMR promoter. A, line diagram showing binding of MTA1 to the human HMMR promoter. To carry out ChIP-based promoter walk analysis, the human HMMR promoter was divided into six regions. ...

Scanning this region for the available transcription factors using the AliBaba2.1 program revealed the availability of only one consensus c-Jun-binding site. Furthermore, ChIP analysis with the anti-c-Jun antibody resulted in the recruitment of c-Jun to only region I (−321 to −102) of the HMMR promoter (Fig. 5D). To illustrate the presence of the coactivator complex, we performed double-ChIP analysis. The MTA1·c-Jun complex was recruited to the HMMR promoter (Fig. 5E). In addition, we observed increased HMMR transcription when MTA1 and c-Jun were transfected, along with HMMR promoter activity (Fig. 6A). Furthermore, to demonstrate the direct binding of c-Jun to the HMMR promoter, we next performed EMSA analysis using oligonucleotides encompassing the c-Jun site (−179 to −173 ) using the nuclear extracts prepared from the MCF-7 cells (Fig. 6B). The specificity of the distinct protein·DNA complex was verified by supershift analysis using anti-c-Jun or anti-MTA1 antibody (Fig. 6B). We noticed supershifts with anti-c-Jun and anti-MTA1 antibodies but not with the control IgG antibody (Fig. 6B). Furthermore, to establish the role of c-Jun in the activation of HMMR transcription, we next mutated the consensus c-Jun-binding site in the HMMR promoter and investigated the HMMR promoter activity in presence of c-Jun and MTA1 using this mutant HMMR promoter. We found decreased transcription with the mutant HMMR promoter compared with the wild-type promoter, and furthermore, we did not observe any increase in its transcription even after transfection with c-Jun and MTA1 (Fig. 6C). Together, these results suggest a role of the MTA1·pol II·c-Jun coactivator complex in the regulation of HMMR transcription.

FIGURE 6.
MTA1·c-Jun coactivator complex activates HMMR transcription. A, lower panel, HMMR promoter-luciferase (HMMR Pro-luc) activity after cotransfection with either MTA1 or c-Jun or with both in MCF-7 cells. The luciferase assay was carried out as described ...

MTA1, an Upstream Regulator of HMMR Function

HMMR-mediated motility of cancer cells in response to HA depends upon the accumulation of HMMR. We therefore investigated the levels of HMMR in MCF-7 and MDA-MB-231 human breast cancer cells by indirect immunofluorescence staining with an anti-HMMR antibody. The accumulation of HMMR increased in the MDA-MB-231 cells compared with the MCF-7 cells, and MTA1 depletion led to a drastic reduction in the levels of HMMR (Fig. 7, A and B). These results suggest that MTA1 functions as an upstream modifier of the expression of HMMR and, consequently, its function.

FIGURE 7.
MTA1 is an upstream regulator of HMMR. A, effect of MTA1 knockdown on the cell surface accumulation of HMMR protein in MCF-7 cells (less invasive breast tumor cells) by confocal analysis. B, effect of MTA1 knockdown using MTA1-specific siRNA 2 on the ...

HMMR, a Mediator of MTA1-mediated Invasion and Motility

We found strong evidence for a role of MTA1 in the accumulation of HMMR in breast cancer cell lines. This finding prompted us to investigate the role of HMMR in MTA1-mediated invasion and motility. We found that siRNA-mediated knockdown of HMMR was accompanied by substantial inhibition of cell motility and reduced cell invasiveness of the MDA-MB-231 cells (Fig. 8, A and B). Furthermore, we also evaluated the effect of HMMR knockdown and overexpression on the migration and invasiveness of MCF-7 cells overexpressing MTA1. We found a decreased migration and invasiveness of cells with HMMR knockdown, whereas cells overexpressing both MTA1 and HMMR were more invasive and showed higher migration levels compared with control cells (Fig. 8, C and D). These results demonstrate that HMMR is a mediator of MTA1-mediated invasion and motility. Collectively, these findings allow us to propose that the increased expression of MTA1 in cancer cells activates the expression and accumulation of HMMR, triggering the cell invasion and migration of breast cancer cells.

FIGURE 8.
MTA1 regulates HMMR function. A, left panel, effect of HMMR knockdown on the motility of MDA-MB-231 cells. Right panel, quantitation of the average wound closure. B, left panel, effect of HMMR knockdown on the invasiveness of MDA-MB-231 cells. Right panel ...

In summary, our finding of MTA1 regulation of HMMR has introduced a new regulatory player to the signaling cascade in the event of invasion and migration. HMMR is a novel breast cancer susceptibility gene, and the association of a homozygous variation of this gene and early-onset breast cancer has been identified (40). HMMR is also a tumor-associated antigen found in solid and blood tumors (38). Although there are many reports on the overexpression and involvement of HMMR in cell invasion and migration in different types of advanced cancers (1014), the transcriptional regulation of HMMR expression is poorly understood. In this context, the studies presented here provide a mechanistic basis for the contribution of MTA1 in positively modulating HMMR expression, leading to cell invasion. As MTA1 regulates its target genes by acting either as a transcriptional corepressor or coactivator (2124), our results show that HMMR is a target of MTA1 and that MTA1 enhances HMMR expression at the transcriptional level by directly interacting with the HMMR promoter as a part of the pol II·c-Jun complex. In support of this, we have also observed a strong meta-analysis correlation between MTA1 and HMMR in the tissue samples of both breast cancer and prostate cancer patients.

Our results also suggest that MTA1 affects the expression of both intracellular and extracellular HMMRs (Fig. 3, A–D, and Fig. 7, A and B), promoting cell motility and invasion via interactions with HA and the cell surface (41, 42). Because of its ability to bind to HA, cell surface HMMR activates multiple mitogenic signaling pathways involved in breast cancer progression, including Ras (43), pp60c-src (44), and ERK1/2 (10). Cell surface HMMR is required for the sustained activation and intracellular targeting of ERK1/2 in dermal wound fibroblasts (41), suggesting that extracellular HMMR could potentially function in tumor progression to increase the intensity and duration of signaling pathways associated with tumor invasion/motility. In this study, we have identified the decreased accumulation of extracellular HMMR and reduced cell migration and invasion with the depletion of MTA1 in breast cancer cell lines (Figs. 7 and and88A). We have demonstrated that MTA1 is an upstream coactivator involved in the regulation of HMMR expression, which may be one of the mechanisms that contributes to the metastatic potential of MTA1. Overall, our results strongly suggest that MTA1 regulates the expression and surface accumulation of HMMR, which has a key role in regulating the motility, invasion, and metastatic potential of cancer cells.

Supplementary Material

Supplemental Data:

Acknowledgments

We thank Dr. Kurt Engeland for generously providing the HMMR promoter-luciferase construct and Dr. Lei Wang (Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX) for microarray data mining. We thank Dr. Norman H. Lee for generously providing prostate cancer cell lines.

*This work was supported, in whole or in part, by National Institutes of Health Grant CA98823 (to R. K.) and Shared Instrumentation Grant S10RR025565.

An external file that holds a picture, illustration, etc.
Object name is sbox.jpgThis article contains supplemental Tables 1–3.

4The abbreviations used are:

HA
hyaluronic acid
HMMR
hyaluronan-mediated motility receptor
pol II
RNA polymerase II
DCIS
ductal carcinoma in situ
qPCR
quantitative RT-PCR
MEF
murine embryonic fibroblast.

REFERENCES

1. Entschladen F., Drell T. L., 4th, Lang K., Joseph J., Zaenker K. S. (2004) Tumor-cell migration, invasion, and metastasis: navigation by neurotransmitters. Lancet Oncol. 5, 254–258 [PubMed]
2. Woodhouse E. C., Chuaqui R. F., Liotta L. A. (1997) General mechanisms of metastasis. Cancer 80, 1529–1537 [PubMed]
3. Yeo T. K., Nagy J. A., Yeo K. T., Dvorak H. F., Toole B. P. (1996) Increased hyaluronan at sites of attachment to mesentery by CD44-positive mouse ovarian and breast tumor cells. Am. J. Pathol. 148, 1733–1740 [PubMed]
4. Tammi M. I., Day A. J., Turley E. A. (2002) Hyaluronan and homeostasis: a balancing act. J. Biol. Chem. 277, 4581–4584 [PubMed]
5. Edward M., Gillan C., Micha D., Tammi R. H. (2005) Tumor regulation of fibroblast hyaluronan expression: a mechanism to facilitate tumor growth and invasion. Carcinogenesis 26, 1215–1223 [PubMed]
6. Tzircotis G., Thorne R. F., Isacke C. M. (2005) Chemotaxis toward hyaluronan is dependent on CD44 expression and modulated by cell type variation in CD44-hyaluronan binding. J. Cell Sci. 118, 5119–5128 [PubMed]
7. Udabage L., Brownlee G. R., Nilsson S. K., Brown T. J. (2005) The overexpression of HAS2, Hyal-2, and CD44 is implicated in the invasiveness of breast cancer. Exp. Cell Res. 310, 205–217 [PubMed]
8. Bourguignon L. Y., Singleton P. A., Zhu H., Zhou B. (2002) Hyaluronan promotes signaling interaction between CD44 and the transforming growth factor β receptor I in metastatic breast tumor cells. J. Biol. Chem. 277, 39703–39712 [PubMed]
9. Hamilton S. R., Fard S. F., Paiwand F. F., Tolg C., Veiseh M., Wang C., McCarthy J. B., Bissell M. J., Koropatnick J., Turley E. A. (2007) The hyaluronan receptors CD44 and Rhamm (CD168) form complexes with ERK1,2 that sustain high basal motility in breast cancer cells. J. Biol. Chem. 282, 16667–16680 [PMC free article] [PubMed]
10. Wang C., Thor A. D., Moore D. H., 2nd, Zhao Y., Kerschmann R., Stern R., Watson P. H., Turley E. A. (1998) The overexpression of RHAMM, a hyaluronan-binding protein that regulates ras signaling, correlates with overexpression of mitogen-activated protein kinase and is a significant parameter in breast cancer progression. Clin. Cancer Res. 4, 567–576 [PubMed]
11. Rein D. T., Roehrig K., Schöndorf T., Lazar A., Fleisch M., Niederacher D., Bender H. G., Dall P. (2003) Expression of the hyaluronan receptor RHAMM in endometrial carcinomas suggests a role in tumor progression and metastasis. J. Cancer Res. Clin. Oncol. 129, 161–164 [PubMed]
12. Zlobec I., Terracciano L., Tornillo L., Günthert U., Vuong T., Jass J. R., Lugli A. (2008) Role of RHAMM within the hierarchy of well established prognostic factors in colorectal cancer. Gut 57, 1413–1419 [PubMed]
13. Crainie M., Belch A. R., Mant M. J., Pilarski L. M. (1999) Overexpression of the receptor for hyaluronan-mediated motility (RHAMM) characterizes the malignant clone in multiple myeloma: identification of three distinct RHAMM variants. Blood 93, 1684–1696 [PubMed]
14. Gust K. M., Hofer M. D., Perner S. R., Kim R., Chinnaiyan A. M., Varambally S., Moller P., Rinnab L., Rubin M. A., Greiner J., Schmitt M., Kuefer R., Ringhoffer M. (2009) RHAMM (CD168) is overexpressed at the protein level and may constitute an immunogenic antigen in advanced prostate cancer disease. Neoplasia 11, 956–963 [PMC free article] [PubMed]
15. Yamano Y., Uzawa K., Shinozuka K., Fushimi K., Ishigami T., Nomura H., Ogawara K., Shiiba M., Yokoe H., Tanzawa H. (2008) Hyaluronan-mediated motility: a target in oral squamous cell carcinoma. Int. J. Oncol. 32, 1001–1009 [PubMed]
16. Sohr S., Engeland K. (2008) RHAMM is differentially expressed in the cell cycle and down-regulated by the tumor suppressor p53. Cell Cycle 7, 3448–3460 [PubMed]
17. Tolg C., Hamilton S. R., Morningstar L., Zhang J., Zhang S., Esguerra K. V., Telmer P. G., Luyt L. G., Harrison R., McCarthy J. B., Turley E. A. (2010) RHAMM promotes interphase microtubule instability and mitotic spindle integrity through MEK1/ERK1/2 activity. J. Biol. Chem. 285, 26461–26474 [PMC free article] [PubMed]
18. Hatano H., Shigeishi H., Kudo Y., Higashikawa K., Tobiume K., Takata T., Kamata N. (2011) RHAMM/ERK interaction induces proliferative activities of cementifying fibroma cells through a mechanism based on the CD44-EGFR. Lab. Invest. 91, 379–391 [PubMed]
19. Song L., Rape M. (2010) Regulated degradation of spindle assembly factors by the anaphase-promoting complex. Mol. Cell 38, 369–382 [PMC free article] [PubMed]
20. Manavathi B., Kumar R. (2007) Metastasis tumor antigens, an emerging family of multifaceted master coregulators. J. Biol. Chem. 282, 1529–1533 [PubMed]
21. Balasenthil S., Gururaj A. E., Talukder A. H., Bagheri-Yarmand R., Arrington T., Haas B. J., Braisted J. C., Kim I., Lee N. H., Kumar R. (2007) Identification of Pax5 as a target of MTA1 in B-cell lymphomas. Cancer Res. 67, 7132–7138 [PubMed]
22. Gururaj A. E., Singh R. R., Rayala S. K., Holm C., den Hollander P., Zhang H., Balasenthil S., Talukder A. H., Landberg G., Kumar R. (2006) MTA1, a transcriptional activator of breast cancer amplified sequence 3. Proc. Natl. Acad. Sci. U.S.A. 103, 6670–6675 [PubMed]
23. Molli P. R., Singh R. R., Lee S. W., Kumar R. (2008) MTA1-mediated transcriptional repression of BRCA1 tumor suppressor gene. Oncogene 27, 1971–1980 [PMC free article] [PubMed]
24. Pakala S. B., Reddy S. D., Bui-Nguyen T. M., Rangparia S. S., Bommana A., Kumar R. (2010) MTA1 coregulator regulates LPS response via MyD88-dependent signaling. J. Biol. Chem. 285, 32787–32792 [PMC free article] [PubMed]
25. Kumar R., Wang R. A., Bagheri-Yarmand R. (2003) Emerging roles of MTA family members in human cancers. Semin. Oncol. 30, 30–37 [PubMed]
26. Manavathi B., Singh K., Kumar R. (2007) MTA family of coregulators in nuclear receptor biology and pathology. Nucl. Recept. Signal. 5, e010. [PMC free article] [PubMed]
27. Toh Y., Pencil S. D., Nicolson G. L. (1994) A novel candidate metastasis-associated gene, mta1, differentially expressed in highly metastatic mammary adenocarcinoma cell lines. cDNA cloning, expression, and protein analyses. J. Biol. Chem. 269, 22958–22963 [PubMed]
28. Pakala S. B., Singh K., Reddy S. D., Ohshiro K., Li D. Q., Mishra L., Kumar R. (2011) TGF-β1 signaling targets metastasis-associated protein 1, a new effector in epithelial cells. Oncogene 30, 2230–2241 [PMC free article] [PubMed]
29. Yoo Y. G., Kong G., Lee M. O. (2006) Metastasis-associated protein 1 enhances stability of hypoxia-inducible factor-1α protein by recruiting histone deacetylase 1. EMBO J. 25, 1231–1241 [PubMed]
30. Moon H. E., Cheon H., Chun K. H., Lee S. K., Kim Y. S., Jung B. K., Park J. A., Kim S. H., Jeong J. W., Lee M. S. (2006) Metastasis-associated protein 1 enhances angiogenesis by stabilization of HIF-1α. Oncol. Rep. 16, 929–935 [PubMed]
31. Du B., Yang Z. Y., Zhong X. Y., Fang M., Yan Y. R., Qi G. L., Pan Y. L., Zhou X. L. (2011) Metastasis-associated protein 1 induces VEGF-C and facilitates lymphangiogenesis in colorectal cancer. World J. Gastroenterol. 17, 1219–1226 [PMC free article] [PubMed]
32. Ghanta K. S., Li D. Q., Eswaran J., Kumar R. (2011) Gene profiling of Mta1 identifies novel gene targets and functions. PLoS ONE 6, e17135. [PMC free article] [PubMed]
33. Kumar R., Balasenthil S., Pakala S. B., Rayala S. K., Sahin A. A., Ohshiro K. (2010) Metastasis-associated protein 1 short form stimulates Wnt1 pathway in mammary epithelial and cancer cells. Cancer Res. 70, 6598–6608 [PMC free article] [PubMed]
34. Kumar R., Wang R. A., Mazumdar A., Talukder A. H, Mandal M., Yang Z., Bagheri-Yarmand R., Sahin A., Hortobagyi G., Adam L., Barnes C. J., Vadlamudi R. K. (2002) A naturally occurring MTA1 variant sequesters estrogen receptor-α in the cytoplasm. Nature 418, 654–657 [PubMed]
35. Reddy S. D., Pakala S. B., Ohshiro K., Rayala S. K., Kumar R. (2009) MicroRNA-661, a c/EBPα target, inhibits metastatic tumor antigen 1 and regulates its functions. Cancer Res. 69, 5639–5642 [PMC free article] [PubMed]
36. Ishigami S., Ueno S., Nishizono Y., Matsumoto M., Kurahara H., Arigami T., Uchikado Y., Setoyama T., Arima H., Yoshiaki K., Kijima Y., Kitazono M., Natsugoe S. (2011) Prognostic impact of CD168 expression in gastric cancer. BMC Cancer 11, 106. [PMC free article] [PubMed]
37. Kong Q. Y., Liu J., Chen X. Y., Wang X. W., Sun Y., Li H. (2003) Differential expression patterns of hyaluronan receptors CD44 and RHAMM in transitional cell carcinomas of urinary bladder. Oncol. Rep. 10, 51–55 [PubMed]
38. Greiner J., Ringhoffer M., Taniguchi M., Schmitt A., Kirchner D., Krähn G., Heilmann V., Gschwend J., Bergmann L., Döhner H., Schmitt M. (2002) Receptor for hyaluronan acid-mediated motility (RHAMM) is a new immunogenic leukemia-associated antigen in acute and chronic myeloid leukemia. Exp. Hematol. 30, 1029–1035 [PubMed]
39. Greiner J., Li L., Ringhoffer M., Barth T. F., Giannopoulos K., Guillaume P., Ritter G., Wiesneth M., Döhner H., Schmitt M. (2005) Identification and characterization of epitopes of the receptor for hyaluronic acid-mediated motility (RHAMM/CD168) recognized by CD8+ T cells of HLA-A2-positive patients with acute myeloid leukemia. Blood 106, 938–945 [PubMed]
40. Pujana M. A., Han J. D., Starita L. M., Stevens K. N., Tewari M., Ahn J. S., Rennert G., Moreno V., Kirchhoff T., Gold B., Assmann V., Elshamy W. M., Rual J. F., Levine D., Rozek L. S., Gelman R. S., Gunsalus K. C., Greenberg R. A., Sobhian B., Bertin N., Venkatesan K., Ayivi-Guedehoussou N., Solé X., Hernández P., Lázaro C., Nathanson K. L., Weber B. L., Cusick M. E., Hill D. E., Offit K., Livingston D. M., Gruber S. B., Parvin J. D., Vidal M. (2007) Network modeling links breast cancer susceptibility and centrosome dysfunction. Nat. Genet. 39, 1338–1349 [PubMed]
41. Tolg C., Hamilton S. R., Nakrieko K. A., Kooshesh F., Walton P., McCarthy J. B., Bissell M. J., Turley E. A. (2006) Rhamm/ fibroblasts are defective in CD44-mediated ERK1,2 motogenic signaling, leading to defective skin wound repair. J. Cell Biol. 175, 1017–1028 [PMC free article] [PubMed]
42. Turley E. A., Noble P. W., Bourguignon L. Y. (2002) Signaling properties of hyaluronan receptors. J. Biol. Chem. 277, 4589–4592 [PubMed]
43. Hall C. L., Yang B., Yang X., Zhang S., Turley M., Samuel S., Lange L. A., Wang C., Curpen G. D., Savani R. C., Greenberg A. H., Turley E. A. (1995) Overexpression of the hyaluronan receptor RHAMM is transforming and is also required for H-ras transformation. Cell 82, 19–26 [PubMed]
44. Hall C. L., Lange L. A., Prober D. A., Zhang S., Turley E. A. (1996) pp60c-src is required for cell locomotion regulated by the hyaluronanreceptor RHAMM. Oncogene 13, 2213–2224 [PubMed]

Articles from The Journal of Biological Chemistry are provided here courtesy of American Society for Biochemistry and Molecular Biology