Large isoform of MRJ (DNAJB6) reduces malignant activity of breast cancer

MDA-MB-231 and MDA-MB-435 cells were maintained as described before [9]. Transfected cells were grown in presence of 500 μg/ml G418 (Invitrogen, Carlsbad, CA, USA). Cells of lines MCF10.DCIS.com, MCF10CAcl.a, and MCF10CAcl.d were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 with 5% horse serum, cholera toxin (100 ng/ml), insulin (10 μg/ml), hydrocortisone (500 ng/ml) and epidermal growth factor (EGF; 25 ng/ml). MCF7 was cultured in DMEM/F12 containing 5% horse serum and insulin (10 μg/ml). All cells were maintained at 37°C with 5% carbon dioxide in a humidified atmosphere.

MCF10DCIS.com (locally aggressive) and metastatic variants MCF10CAcl.a and MCF10CAcl.d are isogenic cell lines derived from in vivo passages of MCF10AT (tumorigenic) in nude mice [10, 11]. These cell lines were obtained from the Barbara Ann Karmanos Cancer Institute (Detroit, MI, USA).

MDA-MB-435 was reported to be derived from the pleural metastases of a 46-year-old woman with breast carcinoma. It forms progressively growing tumors that form metastases in the lungs and regional lymph nodes after injection into the mammary fat pads of 5-week-old athymic nude mice [12]. The origin of MDA-MB-435 has been questioned because it expresses melanoma-associated genes and is reported to originate from the M14 melanoma line [13‐15]. Its breast cancer origin was contested based on the fact that it expresses milk proteins [16, 17]. It is also pertinent to note that MDA-MB-435 metastasizes from the mammary fat pad but rarely from the subcutaneous site. In the present study this cell line was used because it metastasizes from the mammary fat pad.

MRJ(L) cDNA was subcloned into mammalian expression vector pIRES2-EGFP (Clontech, Mountain View, CA, USA) to obtain pIRES2-EGFP-MRJ(L). MDA-MB-231 and MDA-MB-435 cells were transfected with pIRES2-EGFP-MRJ(L). We used the Lipofectamine 2000 reagent (Invitrogen). Briefly, 2 × 10⁶ cells were seeded in a 60 mm dish 1 day before transfection. Plasmid (8 to 10 μg) was mixed the Lipofectamine 2000 reagent, as recommended by the manufacturer. The DNA-Lipofectamine 2000 complex (final volume 1 ml) was added to the plate containing 4 ml serum-free DMEM/F12. Twelve hours later the media were replaced with growth medium without any selection. Transfection was monitored by examining the tranfectants for the green fluorescence. Cells were detached approximately 45 hours after transfection. Using FACSVantage SE (Becton Dickinson), the top 20% fluorescent (enhanced green fluorescent protein [EGFP]) cells were selected (correspondingly high MRJ [L] expressors because of the internal ribosome entry site [IRES]). The expressor cells were passaged in medium with G418. We used the expressors as a pooled population to avoid any clonal variation. All cell lines were used within 10 to 12 passages to avoid variations.

MRJ(L) with wild-type and mutant nuclear localization signal (NLS) were cloned in frame with a carboxyl-terminal EGFP tag in the vector pEGFP-N1. The wild-type NLS from amino acids 305 to 313 (KRKKQKQR) was mutated to (RPDRPETT) using PCR. The corresponding plasmids are called as pEGFP-N1-MRJ(L)^WT and pEGFP-N1-MRJ(L)^mut.

Cells (2 × 10⁶) were plated in 10 cm plate containing appropriate growth medium overnight. The growth medium was replaced with 3 ml serum-free phenol-free DMEM/F12. The conditioned medium was harvested 18 hours later, spun at 1,300 rpm at 4°C for 10 minutes, and concentrated eightfold using a Millipore Amicon Ultra 4 Centrifugal Filter Unit (Billerica, MA, USA) with a 10 KDa cut-off. The concentrated conditioned media samples were resolved on SDS-PAGE and immunoblotted for proteins of interest.

Plates (10 cm) at 90% confluence were washed twice with calcium and magnesium-free phosphate buffered saline (PBS) and lysed with cold lysis buffer (150 mmol/l NaCl, 50 mmol/l Tris, 1% NP-40, and protease and phosphatase inhibitors). The lysates were kept on ice for 1 hour and centrifuged at 10,000 rpm for 30 minutes at 4°C. Protein concentration was measured using Bradford reagent (BioRad Laboratories, Hercules, CA, USA). Proteins (20 μg) were subjected to SDS-PAGE and transferred to polyvinylidene fluoride membrane (0.2 μm). The membranes were blocked with 5% skimmed milk in Tris-buffered saline Tween-20 (1 mol/l Tris [pH 7.5], 9% NaCl and 0.05% Tween-20) and incubated with primary antibodies overnight at 4°C. After washes in Tris-buffered saline Tween-20 and incubation with the respective horseradish peroxidase-tagged secondary antibody, the blots were developed using SuperSignal™ (Pierce, Rockford, IL, USA). For detection of MRJ, 5% milk in PBS containing 0.2% Tween-20 was used, and for detection of osteopontin, 3% bovine serum albumin in PBS containing 0.05% Tween was used as blocking buffer.

We used the following antibodies (dilutions given in parenthesis): anti-DNAJB6 antibody (1:5,000) from Abnova Corporation (Taipei City, Taiwan); anti-osteopontin (1:2,500) from Sigma (St. Louis, MO, USA); anti-KiSS1 antibody (1:500) from Abcam (Cambridge, MA, USA); anti-osteonectin (1:1,000) from Haematologic Technologies Inc. (Essex Junction, VT, USA); anti-nucleophosmin (1:500) from Abcam; anti-zinc α₂-glycoprotein (1:500); anti-VGF nerve growth factor (1:500) from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA); and β-actin (1:30,000) from BioRad.

Cells were cultured to confluence on six-well plates, which were previously externally marked with parallel lines to use as guides for the subsequent photography. A central linear wound (perpendicular to the guide lines) was made with a 200 μl sterile pipet tip. Media were changed gently to remove any floating cells. Phase micrographs of the wound cultures were taken at 0 and 16 hours (average doubling time is 18 to 20 hours). The photographs were analyzed by measuring the distance from the wound edge of the cell sheet to the original wound site. Migration activity was calculated as the mean distance between edges in 12 fields per well. Each test group was assayed in triplicate, and the results are expressed relative to vector control cell migration.

Migration and invasion assays were conducted using 8 μm polyethylene terpthalate filters (BD Pharmingen, San Diego, CA, USA), as described previously [18]. Cells migrated to the lower sides of the trans-well were stained using Diff-Quik^® reagent (Diff Quik is from International Medical Equipment Inc., San Marcos, CA, USA) and the cell number was counted under a microscope. Each test group was assayed in triplicate. Four different fields of each insert were photographed at 10× magnification using a Zeiss Axiocam 200 M microscope (Zeiss Axiocam : Carl Zeiss Microimaging Inc. Thornwood, NY, USA.). Each field was divided into quadrants, and cells in diagonally opposite quadrants were counted.

Cells (2 × 10³) suspended in 0.35% agar were plated onto a layer of 0.75% bactoagar in DMEM/F12 (5% fetal bovine serum) in six-well tissue culture dishes. Visible colonies (> 50 cells) were counted after 15 days with the aid of a dissecting microscope [9].

Nuclear protein extraction was performed as described previously [19]. Briefly, a confluent monolayer of cells was washed twice in ice-cold PBS. Buffer A (10 mmol/l Hepes [pH 7.9], 10 mmol/l KCl, 0.1 mol/l EDTA, 200 μl of 10% IGEPAL, 1 mmol/l DTT, and protease inhibitor cocktail) was added to the monolayer and the plate was maintained at room temperature for 10 minutes. The lysate was spun at 15,000 g for 3 minutes at 4°C. The supernatant was saved as cytosolic fraction. The pellet was suspended in buffer B (20 mmol/l Hepes [pH 7.9], 0.4 mol/l NaCl, 1 mmol/l EDTA, 10% glycerol, 1 mmol/l DTT, and protease inhibitor cocktail) and mixed vigorously for 2 hours at 4°C. The nuclear lysate was obtained by centrifuging at 15,000 g for 5 minutes at 4°C.

COS7 or MDA-MB-231 cells were transfected with pEGFP-N1-MRJ(L)^WT or pEGFP-N1-MRJ(L)^mut using Lipofectamine 2000 (Invitrogen), in accordance with the manufacturer's instructions. Cells were visualized 32 hours after transfection using Leica Microsystems TCS SP2 confocal microscope with 63× water immersion objective (Leica Microsystems Inc.Bannockburn, IL USA). Leica Confocal Software version 2.61 was used for data analysis and fluorescence/differential interference contrast image overlays.

Cells (10⁷ cells/ml) were re-suspended in ice-cold Hanks balanced salt solution and 0.1 ml was injected into exposed axillary mammary fat pads of anesthetized (ketamine 80 mg/kg, xylazine 14 mg/kg), 6-week-old, female athymic mice (Harlan Sprague-Dawley, Indianapolis, IN, USA). Tumor size was measured weekly and mean tumor diameter was calculated as previously reported [9]. The tumor growth was followed for a period of 4 weeks.

The experimental metastasis assay was performed by injecting 2.5 × 10⁵ MDA-MB-231 cells (in 0.2 ml Hanks balanced salt solution) into the lateral tail vein of female athymic mice aged 3 to 4 weeks, as previously described [9]. After a period of 6 weeks, mice were killed; lungs were removed, rinsed with PBS, and fixed in diluted Bouin's solution (20% Bouin's fixative in neutral buffered formalin) before quantification of surface metastasis. For all of the experiments into tumor growth and metastasis, eight mice were used per group and each experiment was repeated once. Animals were maintained under the guidelines of the National Institutes of Health and Institutional Animal Care and Use Committee (IACUC) of the University of South Alabama, Mobile. Food and water were provided ad libitum.

The analysis of secreted proteome was conducted as previously described [20]. Briefly, proteins from the conditioned serum-free medium were concentrated using a tC2 reversed-phase Sorbent column. The protein sample in 0.1% trifluoroacetic acid was loaded onto the cartridge and washed with 0.1% trifluoroacetic acid. Proteins were eluted with increasing amounts of acetonitrile in 0.1% trifluoroacetic acid from 30% to 70% at 0.1 ml/minute, concentrated to dryness, and digested with trypsin. The resulting peptides were analyzed by liquid chromatography-mass spectrometry. Electrospray tandem mass spectrometry was performed with a Q-TOF Ultima API-US mass spectrometer (Waters, Milford, MA, USA) equipped with a nanoflow electrospray.

The resulting data files were searched using an in-house MASCOT search engine (version 2.1.03; Matrix Science Ltd, London, UK) [21]. Ion scores higher than 35 (P < 0.05) were considered significant. Only proteins matching at least two peptides in MASCOT were accepted.

TRIzol reagent (Invitrogen) was used to isolate total RNA from cultured cells. RNA was treated with DNase I (Promega, Madison, WI, USA). cDNA synthesis was carried out using a cDNA synthesis kit (Applied Biosystems Inc., Foster City, CA, USA) using 1 μg total RNA as the template and random primers. Normal breast RNA was purchased from Clontech. Real-time quantitative RT-PCR analysis was performed on the experimental mRNAs. The PCR primers and probes for KiSS1, nucleophosmin (NPM1), osteonectin (SPARC), osteopontin (SPP1), zinc binding α₂-glycoprotein 1 (AZGP1) and VGF nerve growth factor inducible (VGF), and endorse control gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Applied Biosystems Inc. Quantative RT-PCR was performed on an ABI 7500 HT instrument (Applied Biosystems, Foster City, CA, USA). The gene expression Δ CT values of mRNAs from each sample were calculated by normalizing with internal control GAPDH and relative quantitation values were plotted using SDS software v1.2 (Applied Biosystems Inc.). Real-time quantitative RT-PCR analysis was performed on the experimental mRNAs in triplicate, and the experiment was repeated once from an independent passage to confirm the findings.

Breast carcinoma progression array (CC08-00-001) developed by Cybrdi Inc. (Frederick, MA, USA) was probed by Cybrdi Inc. The arrays were stained by 1:100 dilution (10 μg/ml) of DNAJB6 monoclonal antibody. Mouse IgG₁ from Vector PK-6102 (Vector Laboratories, Burlingame, CA, USA) was used for the isotype background control. Photomicrographs were taken in the area of most intense and diffuse staining for MRJ. The intensity of staining of tumor cells was assessed as 0 (no staining) to 3 (strongest possible intensity of staining). The immunoscore was derived as the product of the percentage of cells at each intensity and the corresponding intensity [18]. The products were added to obtain an average immunoscore for the group.

The animal experiments were performed under protocol #06042, approved by the IACUC committee of the University of South Alabama, Mobile.

We assessed the expression of MRJ(L) in breast cancer cell lines using a real-time quatitative RT-PCR. As seen in Figure 1a, all breast cancer cell lines tested by us express very low levels of MRJ(L) compared with the expression seen in normal breast. However, among the cancer cell lines, MDA-MB-231 expressed MRJ(L) at a relatively high level.

We validated this observation at the protein level by an immunoblot for MRJ. As seen in Figure 1b, all cell lines exhibited a clearly detectable level of MRJ(S) (28 kDa). Corresponding to the quatitative RT-PCR data, the larger isoform MRJ(L) (38.6 kDa) was not detectable. Upon prolonged exposure, MDA-MB-231 cells exhibited weakly detectable MRJ(L).

We probed a breast carcinoma progression tissue microarray using antibody specific to MRJ. The antibodies currently available are unable to distinguish between small and large isoforms. The results showed that 80% of the normal breast cases (four out of five) were positive for MRJ staining. We also found that among benign cases nearly half of the tissues stained positive (48%), whereas in infiltrating ductal carcinoma (IDC) cases (grades I and II) 50% cases stained positive (six out of 12 cases). Importantly, only one out of 6 cases (17%) of IDC grade III stained positive, whereas all six cases of IDC with lymph node metastasis exhibited complete absence of staining. Table 1 also outlines the average immunoscores, which decrease with aggressiveness of the tumor. Figure 1c shows intense nuclear and cytoplasmic staining in cystic hyperplasia (panel 1). In contrast, this staining is absent in IDC grade III (panel 2).

The full length DNAJB6 isoform a (MRJ [L]) is comprised of 326 amino acids, whereas the shorter isoform b, (MRJ(S)), has 242 amino acids. MRJ(S) lacks the carboxyl-terminal 95 amino acids of MRJ(L) but contains an additional 10 amino acids (KEQLLRLDNK). Apart from the differences at the carboxyl-terminus, both isoforms share identical structure, containing a conserved J domain (70 amino acids) and a glycine/phenylalanine domain (Figure 2a) [22].

PSORT II software (PSORT II is based on the collaboration of Kenta Nakai, Ph.D.(Human Genome Center, Institute for Medical Science, University ot Tokyo, Japan), with Paul Horton (National Institute of Advanced Industrial Science and Tachnology, Tokyo, Japan.,) [23] predicted the presence of an NLS in MRJ(L) from amino acids 305 to 320 (KRKKQKQREESKKKK). We constructed an in-frame fusion of the NLS using requisite primers and PCR, and generated a mutant NLS by inserting point mutations. The mutated NLS sequence is RPDRPETTEESKKKK. Both the wild-type and mutated NLSs were cloned into the pEGFP-N1 plasmid to generate an in-frame EGFP fusion. Upon transfection of COS7 cells with these constructs, we see that the wild-type fusion protein MRJ(L)^WT-EGFP is predominantly localized to the nucleus, whereas the mutant NLS protein MRJ(L)^mut-EGFP exhibits a uniform distribution throughout the cell (Figure 2b). Similar results were observed when the MRJ(L)^WT-EGFP and the MRJ(L)^mut-EGFP fusion proteins were tested in breast cancer cell line MDA-MB-231. The analysis of nuclear and cytoplasmic protein fractions obtained from cells ectopically expressing MRJ(L) (stable transfectants are described in the following section) also reveals nuclear as well as cytoplasmic localization of MRJ(L) in MDA-MB-231 and MDA-MB-435 cells. However, MRJ(S) was found to be almost exclusively cytoplasmic (Figure 2c).

MDA-MB-435 and MDA-MB-231 cells were stably transfected with MRJ(L)-pIRES2-EGFP as well as with the empty vector. The transfectants were subjected to fluorescence-activated cell sorting to collect cells with greater fluorescence intensity (top 20%). Because of the presence of an IRES, the high fluorescence corresponds to high level of expression of MRJ(L). This population shorted via fluorescence-activated cell sorting was expanded by propagation using G418 resistance and used for subsequent experiments. The expression of MRJ(L) was confirmed by Western blot analysis (Figure 3a).

The in vitro attributes of tumor progression were studied using wound healing assay, invasion, migration and soft agar colonization. MRJ(L) expressors of MDA-MB-231 exhibited 50% motility in a wound healing assay compared with the vector control (Figure 3b). The trend was same for MRJ(L) expressors of MDA-MB-435 MRJ(L), which exhibited 30% motility (Figure 3b). Expressors of MDA-MB-435 showed a highly reduced (10% of vector control) capacity for anchorage-independent growth when tested by colony formation ability in soft agar. Moreover, the colonies that formed in MDA-MB-435-MRJ(L) were small and grew much slower than did the vector control (Figure 3c). The MDA-MB-231 cells did not exhibit a significant change in the soft agar colonization, although the overall trend appeared to be lower than vector control (data not shown). Additionally MRJ(L) expressors of MDA-MB-231 exhibited reduced capacity to migrate (40% of the vector control) and invade (60% of the vector control) through Matrigel™ (BD biosciences San Jose, CA, USA) (Figure 3d, e). The MRJ(L) expressors of MDA-MB-435 also exhibited reduced migration (60% of the vector control) but showed a modest decrease in invasion (85% of the vector control; Figure 3d, e).

The MRJ(L) expressing MDA-MB-231 and MDA-MB-435 and the corresponding empty vector transfectants were independently assayed for orthotopic (mammary fat pad) tumor growth in nude mice. The tumor growth of MRJ(L) expressors of both the cell lines were found to be retarded when compared with the empty vector control (Figure 4a, b).

The MRJ(L) expressing MDA-MB-231 cells exhibited a significantly decreased (P < 0.05) ability to establish pulmonary metastases upon tail vein injection into athymic mice. The MRJ(L) expressing cells showed 65% fewer lung metastases compared with the vector control cells (Figure 4d).

Conditioned cell-free and serum-free media from MRJ(L) expressing MDA-MB-435 were compared with the corresponding vector control. Our results (Table 2) show that SPP1, SPARC, AZGP1, VGF, and NPM1 were notably downregulated in MRJ(L) expressors. Conversely, we also found that the metastasis suppressor KiSS1 was upregulated in the secreted proteome of MRJ(L)-expressing cells compared with controls. Analysis of the serum-free medium by immunoblot analysis confirmed that levels of SPP1, SPARC, VGF, NPM1, and AZGP1 were below the limit of detection in MDA-MB-435-MRJ(L) culture medium (Figure 5a). The increased expression of KiSS1 in MRJ(L) expressors was confirmed by analyzing the cell lysate (Figure 5a). We found that MDA-MB-231 does not secrete SPP1 [24] and SPARC [25], which is in agreement with previously reported findings. Also, it does not secrete VGF and AZGP (data not shown). However, Western blot analysis revealed increased level of KiSS1 expression and decreased level of NPM1 in MRJ(L) expressing MDA-MB-231.

We compared the expression levels of SPP1, AZGP1, VGF, SPARC, NPM1, and KiSS1 using quantitative RT-PCR to determine whether the change in the levels of secreted proteome was due to change in transcription. Real-time PCR comparison demonstrated reduced expression of SPP1 (17-fold), AZGP (10-fold), VGF (9.4-fold), SPARC (7-fold) and NPM1 (1.3-fold), and increased expression of KiSS1 (13-fold) in MDA-MB-435-MRJ(L) cells compared with the vector control (Figure 5b).