Introduction Mammalian relative of DnaJ (MRJ [DNAJB6]), a novel member of the human DnaJ family, has two isoforms. was ectopically expressed in two breast cancer cell lines. These cell lines were evaluated for their in vitro correlates of tumor aggressiveness, such as invasion, migration, and anchorage independence. The cell lines were also evaluated for in vivo tumor growth and metastasis. The secreted proteome of the MRJ(L) expressors was analyzed to elucidate the biochemical changes brought about by re-expression of MRJ(L). Results We found that MRJ(L) is expressed at a significantly lower level in aggressive breast cancer cell lines compared with normal breast. Furthermore, in clinical cases of breast cancer expression of MRJ is lost as the grade of infiltrating ductal carcinoma advances. Importantly, MRJ staining is lost in those cases that also had lymph node metastasis. We report that MRJ(L) is a protein with a functional nuclear localization sequence. Expression of MRJ(L) via an exogenous promoter in breast cancer cell line MDA-MB-231 and in MDA-MB-435 (a cell line that metastasizes from the mammary fat pad) decreases their migration and invasion, reduces their motility, and significantly reduces orthotopic tumor growth in nude mice. Moreover, the secreted proteome of the MRJ(L)-expressing cells exhibited reduced levels of tumor progression and metastasis promoting secreted proteins, such as SPP1 (osteopontin), AZGP1 (zinc binding 2-glycoprotein 1), SPARC (osteonectin), NPM1 (nucleophosmin) and VGF (VGF nerve growth factor inducible). On the other hand, levels of the secreted metastasis-suppressor KiSS1 (melanoma metastasis suppressor) were increased in the secreted proteome of the MRJ(L)-expressing cells. We confirmed by quantitative RT-PCR analysis that the secreted profile reflected altered transcription of the respective genes. Conclusion Collectively, our data indicate an important role for a totally uncharacterized isoform of DNAJB6 in breast cancer. We show that MRJ(L) is a nuclear protein that Rabbit Polyclonal to RFX2 is lost in breast cancer, that regulates several key players in tumor formation and metastasis, and that is functionally able to retard tumor growth. Introduction Mammalian relative of DnaJ (MRJ) is a class II DnaJ/heat shock protein (Hsp)40 family protein [1,2]. The human MRJ gene has been mapped to chromosome 7q36.3 and encodes two spliced variants (reported in GenBank) [3]: isoform a (referred to here as MRJ [L]) and isoform b (MRJ [S]). Murine MRJ has been shown to be essential for murine placental development [4]. MRJ(S) has also been implicated as an important chaperone in Huntington’s disease [5,6]. Over-expressed MRJ(S) effectively suppressed polyglutamine-dependent protein aggregation, caspase activity, and cellular toxicity in neurons. MRJ(S) specifically has also been shown to regulate keratin-8/18 filament organization [7]. Interesting recent studies have shown a role for MRJ(S) in blocking calcineurin-induced cardiomyocyte buy GnRH Associated Peptide (GAP) (1-13), human hypertrophy [8]. All studies of MRJ have focused on the shorter isoform (isoform b, 242 amino acids). Our study elucidates the role of the longer isoform (isoform a, 326 amino acids) in breast cancer cells. Here, we report for the first time that MRJ(L) levels are almost undetectable in aggressive breast cancer cells and in advanced grade infiltrating ductal carcinoma. MRJ(L) localizes to the nucleus and suppresses tumorigenicity and metastasis of breast cancer cells. Furthermore, cells expressing MRJ(L) buy GnRH Associated Peptide (GAP) (1-13), human show an altered profile of the secreted proteome. Several secreted proteins that are known to play important roles in promoting cancer progression are downregulated. This is concomitant with upregulation of a breast and melanoma metastasis suppressor protein, namely KiSS1 (melanoma metastasis suppressor; metastin). Materials and methods Cell culture MDA-MB-231 and MDA-MB-435 cells were maintained as described before [9]. Transfected cells were grown in presence of 500 g/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 g/ml), hydrocortisone (500 ng/ml) and epidermal growth factor (EGF; 25 ng/ml). MCF7 was cultured in DMEM/F12 containing 5% horse buy GnRH Associated Peptide (GAP) (1-13), human serum and insulin (10 g/ml). All cells were maintained at 37C 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.