Experimental evidence indicates that prostate apoptosis response-4 (Par-4, also known as PAWR) is a key regulator of cancer cell survival and may be a target for cancer-selective targeted therapeutics. Par-4 was first identified in prostate cancer cells undergoing apoptosis. Both intracellular and extracellular Par-4 have been implicated in apoptosis. Relatively little is known about the role of Par-4 in breast cancer cell apoptosis. In this study, we sought to investigate the effects of Par-4 expression on cell proliferation, apoptosis and drug sensitivity in breast cancer cells. MCF-7 cells were stably transfected with expression vectors for Par-4, or transiently transfected with siRNA for Par-4 knockdown. Cell proliferation assays were performed using MTT and apoptosis was evaluated using acridine orange staining, fluorescence microscopy and flow cytometry. Par-4 overexpression reduced MCF-7 proliferation rates. Conversely, Par-4 knockdown led to increased MCF-7 proliferation. Par-4 downregulation also led to increased BCL-2 and reduced BID expression. Par-4 overexpression did not affect the cell cycle profile. However, MCF-7 cells with increased Par-4 expression showed reduced ERK phosphorylation, suggesting that the inhibition of cell proliferation promoted by Par-4 may be mediated by the MAPK/ERK1/2 pathway. MCF-7 cells with increased Par-4 expression showed a marginal increase in early apoptotic cells. Importantly, we found that Par-4 expression modulates apoptosis in response to docetaxel in MCF7 breast cancer cells. Par-4 exerts growth inhibitory effects on breast cancer cells and chemosensitizes them to docetaxel.
PAR-4 is a tumor suppressor protein with a pro-apoptotic function and down-regulation of PAR-4 is seen in a variety of tumors. PHLDA1 gene overexpression has been shown to reduce cell proliferation and induce cell death in a variety of cell types. In this study, 229 cases of oral squamous cell carcinoma (OSCC), arranged in a tissue microarray, were analyzed by immunohistochemistry. PAR-4 expression was predominantly moderate to strong and expression of PHLDA1 was predominantly negative or weak. Cytoplasmic expression of PAR-4 was associated with advanced clinical stage. Expression of PHLDA1 was associated with advanced clinical stage of the tumour. Five-year overall and disease-free survival rates differed significantly between cases that did and cases that did not express PHLDA1, and by multivariate analysis, expression of PHLDA1 and PAR-4 were independent prognostic factors in OSCC patients. Expression of PAR-4 and PHLDA1 is altered in OSCC and might be a valuable prognostic indicator for this disease.
ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin motifs) is a member of the ADAMTS family of metalloproteases. Here, we investigated mRNA and protein levels of ADAMTS-1 in normal and neoplastic tissues using qPCR, immunohistochemistry and immunoblot analyses, and we addressed the role of ADAMTS-1 in regulating migration, invasion and invadopodia formation in breast tumor cell lines.
The PKC apoptosis WT1 regulator gene, also named prostate apoptosis response-4 (PAR-4), encodes a pro-apoptotic protein that sensitizes cells to numerous apoptotic stimuli. Insulin-like growth factor-1 (IGF-1) and 17?-estradiol (E2), two important factors for breast cancer development and progression, have been shown to down-regulate PAR-4 expression and inhibit apoptosis induced by PAR-4 in neuronal cells. In this study, we sought to investigate the mechanisms of regulation of PAR-4 gene expression in MCF-7 cells treated with E2 or IGF-1. E2 (10 nM) and IGF-1 (12.5 nM) each down-regulated PAR-4 expression in MCF-7 cells after 24 h of treatment. The effect of E2 was dependent on ER activation, as demonstrated by an increase in PAR-4 expression when cells were pretreated for 1 h with 1 µM ICI-182,780 (ICI) before receiving E2 plus ICI. The effect of IGF-1 was abolished by pre-treatment for 1 h with 30 µM LY294002 (a specific PI3-K inhibitor), and significantly inhibited by 30 µM SB202190 (a specific p38MAPK inhibitor). We also demonstrated that E2 acts synergistically with IGF-1, resulting in greater down-regulation of PAR-4 mRNA expression compared with E2 or IGF-1 alone. Our results show for the first time that E2 and IGF-1 inhibit PAR-4 gene expression in MCF-7 cells, suggesting that this down-regulation may provide a selective advantage for breast cancer cell survival.
Substantial experimental evidence indicates that PAWR gene (PKC apoptosis WT1 regulator; also named PAR-4, prostate apoptosis response-4) is a central player in cancer cell survival and a potential target for cancer-selective targeted therapeutics. However, little is known about the role of PAR-4 in breast cancer. We investigated the possible role of PAR-4 expression in breast cancer. IHC results on tissue microarrays containing 1,161 primary breast tumor samples showed that 57% (571/995) of analyzable cases were negative for PAR-4 nuclear staining. Down-regulation of nuclear PAR-4 protein expression predicted a poor prognosis for breast cancer patients (OS; P=0.041, log-rank test). PAR-4 down-regulation also correlates with poor survival in the group of patients with luminal A subtype breast cancer (P=0.028). Additionally, in this large series of breast cancer patients, we show that ERBB2/HER2, EGFR and pAKT protein expression are significantly associated with shorter disease-free survival and overall survival, but the prognosis was even worse for HER2-positive, EGFR-positive or pAKT-positive breast cancer patients with tumors negative for nuclear PAR-4 expression. Furthermore, using three-dimensional (3D) cell culture we provide preliminary results showing that PAR-4 is highly expressed in the MCF10A cells inside the acini structure, suggesting that PAR-4 might have a role in the lumen acini formation. Taken together, our results provide, for the first time, evidence that PAR-4 may have a role in the process of the mammary gland morphogenesis and its functional inactivation is associated with tumor aggressive phenotype and might represent an additional prognostic and predictive marker for breast cancer.
An increasing number of studies have shown altered expression of secreted protein acidic and rich in cysteine (SPARC) and N-myc down-regulated gene (NDRG1) in several malignancies, including breast carcinoma; however, the role of these potential biomarkers in tumor development and progression is controversial. In this study, NDRG1 and SPARC protein expression was evaluated by immunohistochemistry on tissue microarrays containing breast tumor specimens from patients with 10 years of follow-up. NDRG1 and SPARC protein expression was determined in 596 patients along with other prognostic markers, such as ER, PR, and HER2. The status of NDRG1 and SPARC protein expression was correlated with prognostic variables and patient clinical outcome. Immunostaining revealed that 272 of the 596 cases (45.6%) were positive for NDRG1 and 431 (72.3%) were positive for SPARC. Statistically significant differences were found between the presence of SPARC and NDRG1 protein expression and standard clinicopathological variables. Kaplan-Meier analysis showed that NDRG1 positivity was directly associated with shorter disease-free survival (DFS, P < 0.001) and overall survival (OS, P < 0.001). In contrast, patients expressing low levels of SPARC protein had worse DFS (P = 0.001) and OS (P = 0.001) compared to those expressing high levels. Combined analysis of the two markers indicated that DFS (P < 0.001) and OS rates (P < 0.001) were lowest for patients with NDRG1-positive and SPARC-negative tumors. Furthermore, NDRG1 over-expression and SPARC down-regulation correlated with poor prognosis in patients with luminal A or triple-negative subtype breast cancer. On multivariate analysis using a Cox proportional hazards model, NDRG1 and SPARC protein expression were independent prognostic factors for both DFS and OS of breast cancer patients. These data indicate that NDRG1 over-expression and SPARC down-regulation could play important roles in breast cancer progression and serve as useful biomarkers to better define breast cancer prognosis.
The phosphatidylinositol 3-kinase/AKT axis is an important cell-signaling pathway that mediates cell proliferation and survival, two biological processes that regulate malignant cell growth. The phosphatidylinositol 3-kinase CA gene encodes the p110? subunit of the phosphatidylinositol 3-kinase protein. There are phosphatidylinositol 3-kinase CA mutations in several types of human tumors, and they are frequently observed in breast cancer. However, these mutations have not been investigated in Brazilian breast cancer patients.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.