Mitra Aparna, Fillmore Rebecca A, Metge Brandon J, Rajesh Mathur, Xi Yaguang, King Judy, Ju Jingfang, Pannell Lewis, Shevde Lalita A, Samant Rajeev S
Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, N University Blvd, Mobile, Alabama 36688, USA.
Breast Cancer Res. 2008;10(2):R22. doi: 10.1186/bcr1874. Epub 2008 Mar 7.
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.
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).
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.
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.
DnaJ的哺乳动物同源物（MRJ [DNAJB6]）是人类DnaJ家族的一个新成员，有两种异构体。较小的异构体MRJ(S)，主要因其在亨廷顿舞蹈病中可能发挥的作用而被研究。关于较长异构体MRJ(L)的任何生物学活性尚无报道。我们研究了该分子在乳腺癌中是否发挥作用。我们在乳腺癌细胞系和乳腺癌组织微阵列中对MRJ的表达进行了有趣的观察，从而推动了我们的研究，如下所述。
使用实时PCR评估几种乳腺癌细胞系中MRJ(L)的表达。使用蛋白质免疫印迹分析研究乳腺癌细胞系中小异构体和大异构体的相对水平。用抗MRJ抗体检测乳腺癌进展组织微阵列。MRJ(L)在两种乳腺癌细胞系中异位表达。对这些细胞系进行体外肿瘤侵袭性相关指标评估，如侵袭、迁移和锚定非依赖性。还对细胞系进行体内肿瘤生长和转移评估。分析MRJ(L)表达细胞的分泌蛋白质组，以阐明MRJ(L)重新表达所带来的生化变化。
我们发现，与正常乳腺相比，MRJ(L)在侵袭性乳腺癌细胞系中的表达水平显著降低。此外，在乳腺癌临床病例中，随着浸润性导管癌分级的升高，MRJ的表达缺失。重要的是，在那些也有淋巴结转移的病例中，MRJ染色缺失。我们报告MRJ(L)是一种具有功能性核定位序列的蛋白质。通过外源性启动子在乳腺癌细胞系MDA-MB-231和MDA-MB-435（一种从乳腺脂肪垫转移的细胞系）中表达MRJ(L)，可降低其迁移和侵袭能力，降低其运动性，并显著减少裸鼠原位肿瘤生长。此外，表达MRJ(L)的细胞的分泌蛋白质组中促进肿瘤进展和转移的分泌蛋白水平降低，如骨桥蛋白（SPP1）、锌结合α2糖蛋白1（AZGP1）、骨连接蛋白（SPARC）、核磷蛋白（NPM1）和VGF神经生长因子诱导蛋白（VGF）。另一方面，表达MRJ(L)的细胞的分泌蛋白质组中分泌的转移抑制因子KiSS1（黑色素瘤转移抑制因子）水平升高。我们通过定量RT-PCR分析证实，分泌谱反映了相应基因转录的改变。
总体而言，我们的数据表明DNAJB6的一种完全未被表征的异构体在乳腺癌中起重要作用。我们表明MRJ(L)是一种在乳腺癌中缺失的核蛋白，它调节肿瘤形成和转移中的几个关键因子，并且在功能上能够延缓肿瘤生长。
