The Correlation And Mechanism Between NDRG1 Gene And The Biological Behaviour Of Breast Cancer

The incidence and mortality of breast cancer are increasing each year, which is one of the major cancers causing death of women. Among various prognosticators of breast cancer, disease recurrence and metastasis are key factors affecting the outcome of initial breast cancer therapy. Identification of sensitive biomarkers for metastatic potential and their regulatory genes in breast cancer cells, therefore, is important for both early clinical detection and effective prophylactic therapy.Human NDRG1 (N-myc Downstream Regulated Gene 1) is a metastasis-related gene recently and independently recognized by five research groups. NDRG1 is located on chromosome 8q24.2, with a cDNA length of 1,182 bp that code for a 43 kDa protein. Most published reports have addressed the effects of NDRG1 expression on proliferation, infiltration and metastasis of cells in bladder, colon, and prostate cancers. The exact role NDRG1 plays in cancer metastasis is, however, still largely unclear. Specifically, there has been no report on the relation and mechanism between NDRG1 gene expression and the biological behavior of breast cancer in either Chinese literature or elsewhere.Using three experimental models - resected clinical specimens of human breast cancer, established breast cancer cell lines, and tumor xenograft in nude mouse breast pad - this study investigates the impact of NDRG1 expression on the metastatic potential of breast cancer in an effort to identify NDRG1 as a biomarker predicting early metastasis and a potential target for therapy.Part I Correlation of NDRG1 expression with metastatic potential of human breast cancer in resected clinical specimens and established cell linesPurpose: To see if any correlation exists between NDRG1 expression and metastatic potential of human breast cancer using surgically resected specimens and established cell lines.Methods: NDRG1 protein was detected by immunohistochemistry (IHC), and NDRG1 mRNA was detected by real time reverse transcriptase-polymerase chain reaction (real time RT-PCR) in fresh and paraffin-embedded breast cancer specimens and breast cancer cell lines with different metastatic potentials.Results: The amounts of NDRG1 protein and mRNA found in breast cancer patients with lymph node metastases were 47.7% and 20.33% of that found in comparable patients with disease localized to their breasts, respectively (p < 0.01). The amount of NDRG1 mRNA detected in MCF7 (a relatively non-invasive breast cancer cell line with low metastatic potential) was 10.8 times that of MDA-MB-231 (an invasive breast cancer cell line with high metastatic potential) (p < 0.01).Conclusions: NDRG1 expression (transcription and translation) is negatively correlated with the metastatic potential of human breast cancer, suggesting that NDRG1 may serve as a molecular biological marker for risk of breast cancer metastasis.Part II Effects of NDRG1 overexpression on proliferation, invasiveness, and metastasis of breast cancer cell line MDA-MB-231 in vitro and in vivoPurpose: To investigate the effects of NDRG1 overexpression on proliferation, invasiveness, and metastasis of breast cancer cell line MDA-MB-231 in vitro and in vivo.Methods: Lipofectamine^? 2000 was used to stably transfect recombinant plasmids of pEGFP-NDRGl-N3 into human breast cancer cell line MDA-MB-231 known to be highly invasive with high metastatic potential. NDGR1 overexpression in the positive clones was determined by RT-PCR, Western blot analysis, and immunofluorescence. Proliferation of MDA-MB-231 was measured by bromodeoxyuridine (BrdU) incorporation, and the effect on cell cycle distribution was determined by fluorescence-activated cell sorting (FACS). We also investigated the ability of this cell line with transfection-mediated NDRG1 overexpression to invade reconstituted Matrigel and to migrate through polycarbonate filters in transwell chambers. The growth and metastatic potential of these clones were also examined in vivo. We inoculated them into the mammary fat pad of nude mice and observe the growth of xenografts and the appearance of local invasiveness and metastases.Results: Stable cell clones with marked overexpression of NDRG1 mRNA and protein were established. Proliferation, as measured by BrdU incorporation, declined significantly in an MDA-MB-231 clone overexpressing NDRG1, with an increase in cells at the G₀/G₁ phase and a decrease in cells at the S phase of cell cycle. NDRG1 overexpression significantly reduced invasiveness of MDA-MB-231 cells in Matrigel-coated invasion chambers when compared to negative control or cells transfected with vectors only. However, migration through polycarbonate filters was unaffected with no difference found between the parent and transfected cells. The NDRG1 overexpressing clone showed a reduced growth rate of tumor xenograft and reduced local invasiveness and metastasis in vivo following mammary fat pad injection in nude mice.Conclusions: NDRG1 overexpression reduces breast cancer cell proliferation, invasiveness, and metastatic potential both in vitro and in vivo.Part III Possible mechanisms of how NDRG1 affects breast cancer cell invasiveness and metastasisPurpose: To investigate the molecular regulatory mechanisms of NDRG1 in breast cancer cell invasiveness and metastasis.Methods: The expression of MMP-2 and MMP-9 at mRNA and protein levels were determined by real time RT-PCR and Western blot analysis.Results: Overexpression of NDRG1 significantly reduced the levels of MMP-2 and MMP-9 mRNA and active protein in MDA-MB-231 cells when compared to negative control and cells transfected with vectors only.Conclusions: The suppression of invasiveness and metastatic potential of breast cancer cells by NDRG1 requires transcription, translation and post-translation modification of MMP-2 and MMP-9 genes.