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

INTRODUCTION

Mammalian relative of DnaJ (MRJ [DNAJB6]), a novel member of the human DnaJ family, has two isoforms. The smaller isoform, MRJ(S), is studied mainly for its possible role in Huntington's disease. There are no reports of any biologic activity of the longer isoform, MRJ(L). We investigated whether this molecule plays any role in breast cancer. Our studies were prompted by interesting observations we made regarding the expression of MRJ in breast cancer cell lines and breast cancer tissue microarrays, as described below.

METHODS

Expression of MRJ(L) from several breast cancer cell lines was evaluated using real-time PCR. Relative levels of the small and large isoforms in breast cancer cell lines were studied using Western blot analysis. A breast cancer progression tissue microarray was probed using anti-MRJ antibody. MRJ(L) 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 alpha2-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 is lost in breast cancer, that regulates several key players in tumor formation and metastasis, and that is functionally able to retard tumor growth.