BRCA1 and EZH2 cooperate in regulation of prostate cancer stem cell phenotype

Prostate cancer (PCa) is the second most common malignancy and the sixth leading cause of cancer-related death among men worldwide. Prostate carcinogenesis is driven by the accumulation of genetic and epigenetic aberrations, which regulate cancer cell transition between a stem- and nonstem-cell state and accelerate tumor evolution. Elevated expression of enhancer of zeste homolog 2 (EZH2) histone methyltransferase, a core member of the polycomb repressive complex 2 (PRC2), results in cancer progression through histone methylation-driven tumor cells dedifferentiation. Previous studies demonstrated that tumor suppressor breast cancer 1 (BRCA1) is a negative regulator of PRC2-dependent H3K27 methylation. Our recent studies revealed that inhibition of EZH2-mediated histone methylation radiosensitizes prostate cancer stem cells (CSCs) population. However, the link between BRCA1 and EZH2 in regulation of prostate CSCs remains elusive. Present study demonstrated that BRCA1 and EZH2 are coregulated in patients’ tumors and PCa cell lines, and cooperate in regulation of CSC phenotype and properties. Knockdown of BRCA1 expression significantly increases the number and the size of tumor spheres. Inhibition of BRCA1 and EZH2 expression leads to an increase of aldehyde dehydrogenase (ALDH)-positive cell population that is, at least partially, attributed to the upregulation of ALDH1A3 protein. Treatment with a global histone methylation inhibitor 3-Deazaneplanocin A abrogates this regulation, downregulates BRCA1 and EZH2 expression and has an inhibitory effect on the tumorigenic properties of radioresistant PCa cells in vivo. We found that EZH2/BRCA1 signaling mechanisms play an important role in the maintenance of prostate CSC properties and may be a promising target for tumor treatment.     

Triptolide Inhibits Histone Methyltransferase EZH2 and Modulates the Expression of Its Target Genes in Prostate Cancer Cells

The histone methyltransferase EZH2 (enhancer of zeste homolog 2) plays critical roles in prostate cancer(PCa) development and is a potential target for PCa treatment. Triptolide possesses anti-tumor activity, but itis unknown whether its therapeutic effect relates with EZH2 in PCa. Here we described EZH2 as a target forTriptolide in PCa cells. Our data showed that Triptolide suppressed PCa cell growth and reduced the expressionof EZH2. Overexpression of EZH2 attenuated the Triptolide induced cell growth inhibition. Moreover, Triptolidetreatment of PC-3 cells resulted in elevated mRNA levels of target genes (ADRB2, CDH1, CDKN2A andDAB2IP) negatively regulated by EZH2 as well as reduced mRNA levelsan of EZH2 positively regulated gene(cyclin D1). Our findings suggest the PCa cell growth inhibition mediated by Triptolide might be associated withdownregulation of EZH2 expression and the subsequent modulation of target genes.     

Zeste增强子同源物2与肿瘤转移的研究进展

转移是癌症患者死亡和预后不良的主要原因,阐明肿瘤转移机制对改善患者预后至关重要.Zeste增强子同源物2(enhancer of zeste homolog 2,EZH2)是多梳蛋白家族(Polycomb group,PcG)的重要成员,其广泛参与调节多种细胞生理过程,如细胞迁移、细胞周期、细胞增殖、细胞凋亡、DNA损...     

Raf kinase inhibitor protein suppresses nuclear factor-κB-dependent cancer cell invasion through negative regulation of matrix metalloproteinase expression

Accumulating evidence suggests that Raf kinase inhibitor protein (RKIP), which negatively regulates multiple signaling cascades including the Raf and nuclear factor-κB (NF-κB) pathways, functions as a metastasis suppressor. However, the basis for this activity is not clear. We investigated this question in a panel of breast cancer, colon cancer and melanoma cell lines. We found that RKIP negatively regulated the invasion of the different cancer cells through three-dimensional extracellular matrix barriers by controlling the expression of matrix metalloproteinases (MMPs), particularly, MMP-1 and MMP-2. Silencing of RKIP expression resulted in a highly invasive phenotype and dramatically increased levels of MMP-1 and MMP-2 expression, while overexpression of RKIP decreased cancer cell invasion in vitro and metastasis in vivo of murine tumor allografts. Knockdown of MMP-1 or MMP-2 in RKIP-knockdown cells reverted their invasiveness to normal. In contrast, when examining migration of the different cancer cells in a two-dimensional, barrier-less environment, we found that RKIP had either a positive regulatory activity or no activity, but in no case a negative one (as would be expected if RKIP suppressed metastasis at the level of cell migration itself). Therefore, RKIP’s function as a metastasis suppressor appears to arise from its ability to negatively regulate expression of specific MMPs, and thus invasion through barriers, and not from a direct effect on the raw capacity of cells to move. The NF-κB pathway, but not the Raf pathway, appeared to positively control the invasion of breast cancer cells. A regulatory loop involving an opposing relationship between RKIP and the NF-κB pathway may control the level of MMP expression and cell invasion.     

Oncogenic roles of enhancer of zeste homolog 1/2 in hematological malignancies

Polycomb group (PcG) proteins regulate the expression of target genes by modulating histone modifications and are representative epigenetic regulators that maintain the stemness of embryonic and hematopoietic stem cells. Histone methyltransferases enhancer of zeste homolog 1 and 2 (EZH1/2), which are subunits of polycomb repressive complexes (PRC), are recurrently mutated or highly expressed in many hematological malignancies. EZH2 has a dual function in tumorigenesis as an oncogene and tumor suppressor gene, and targeting PRC2, in particular EZH1/2, for anticancer therapy has been extensively developed in the clinical setting. Here, we review the oncogenic function of EZH1/2 and introduce new therapeutic drugs targeting these enzymes.     

Global H3K27 trimethylation and EZH2 abundance in breast tumor subtypes

Polycomb repressive complex 2 (PRC2) and its core member enhancer of zeste homolog 2 (EZH2) mediate the epigenetic gene silencing mark: trimethylation of lysine 27 on histone 3 (H3K27me3). H3K27me3 is characteristic of the chromatin at genes involved in developmental regulation in undifferentiated cells. Overexpression of EZH2 has been found in several cancer types such as breast, prostate, melanoma and bladder cancer. Moreover, overexpression is associated with highly proliferative and aggressive types of breast and prostate tumors. We have analyzed the abundance of EZH2 and H3K27me3 using immunohistochemistry in two large and well-characterized breast tumor data sets encompassing more than 400 tumors. The results have been analyzed in relation to the molecular subtypes of breast tumors (basal-like, luminal A, luminal B, HER2-enriched and normal-like), as well as in subtypes defined by clinical markers (triple negative, ER+/HER2-/Ki67low, ER+/HER2-/Ki67high and HER2+), and were validated in representative breast cancer cell lines by western blot. We found significantly different expression of both EZH2 and H3K27me3 across all subtypes with high abundance of EZH2 in basal-like, triple negative and HER2-enriched tumors, and high H3K27me3 in luminal A, HER2-enriched and normal-like tumors. Intriguingly, the two markers show an inverse correlation, particularly for the basal-like and triple negative tumors. Consequently, high expression of EZH2 was associated with poor distant disease-free survival whereas high expression of H3K27me3 was associated with better survival. Additionally, none of 182 breast tumors was found to carry a previously described EZH2 mutation affecting Tyr641. Our observation that increased expression of EZH2 does not necessarily correlate with increased abundance of H3K27me3 supports the idea that EZH2 can have effects beyond epigenetic silencing of target genes in breast cancer.     

Raf kinase inhibitor protein: a prostate cancer metastasis suppressor gene

Defining the mechanisms that confer metastatic ability on cancer cells is an important goal towards prevention of metastasis. A gene array screen between a non-metastatic prostate cancer cell and its metastatic derivative line revealed decreased expression of Raf kinase inhibitor protein (RKIP) in the metastatic cell line. This finding is consistent with the possibility that loss of RKIP is associated with metastasis. RKIP is expressed in many tissues including brain, lung, and liver. RKIP blocks Raf-induced phosphorylation of MEK. In addition to its modulation of Raf signaling, RKIP modulates both G-protein signaling and NF-kappaB activity. The impact that RKIP has on multiple signaling pathways grants it the ability to play a role in several cellular functions including membrane biosynthesis, spermatogenesis, and neural signaling. Novel cellular functions for RKIP continue to be identified, several of which contribute to cancer biology. For example, RKIP promotes apoptosis of cancer cells, which suggests that loss of RKIP in cancer will protect cancer cells against cell death. Additionally, restoration of RKIP expression ina metastatic prostate cancer cell line does not effect primary tumor growth, but it does inhibit prostate cancer metastasis. These parameters identify RKIP as a metastasis suppressor gene, which suggest that it or proteins it interacts with are putative molecular targets to control metastasis. These findings are supported by the observation that RKIP expression is decreased in metastases of prostate cancer patients, compared to normal prostate or the primary prostate tumor. In this review, RKIP biology and its role in cancer will be described.     

RKIP regulates CCL5 expression to inhibit breast cancer invasion and metastasis by controlling macrophage infiltration

Accumulating evidence suggests that presence of macrophages in the tumor microenvironment add to the invasive and tumor-promoting hallmarks of cancer cells by secreting angiogenic and growth factors. RKIP is a known metastasis suppressor and interferes with several steps of metastasis. However, the mechanistic underpinnings of its function as a broad metastasis suppressor remain poorly understood. Here, we establish a novel pathway for RKIP regulation of metastasis inhibition through the negative regulation of RANTES/CCL5 thereby limiting tumor macrophage infiltration and inhibition of angiogenesis. Using a combination of loss- and gain-of-function approaches, we show that RKIP hinders breast cancer cell invasion by inhibiting expression of the CC chemokine CCL5 in vitro. We also show that the expression levels of RKIP and CCL5 are inversely correlated among clinical human breast cancer samples. Using a mouse allograft breast cancer transplantation model, we highlight that ectopic expression of RKIP significantly decreases tumor vasculature, macrophage infiltration and lung metastases. Mechanistically, we demonstrate that the inhibition of the CCL5 expression is the cause of the observed effects resulting from RKIP expression. Taken together, our results underscore the significance of RKIP as important negative regulator of tumor microenvironment.     

Reduction of Raf-1 kinase inhibitor protein expression correlates with breast cancer metastasis.

PURPOSE: Raf-1 kinase inhibitor protein (RKIP) was originally identified as the first physiologic inhibitor of the Raf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (ERK) pathway. This pathway regulates fundamental cellular functions, including those that are subverted in cancer cells, such as proliferation, transformation, survival, and metastasis. Recently, RKIP has been recognized as a strong candidate for a metastasis suppressor gene in cell and animal model systems. Therefore, we investigated whether RKIP expression is altered in clinical specimens of human primary breast cancers and their lymph node metastases. EXPERIMENTAL DESIGN: Paraffin-embedded tumor samples from 103 breast cancer patients were examined immunohistochemically for the expression of RKIP, activated ERK, and apoptosis. The specificity of the antibodies used was validated by competition experiments with purified recombinant RKIP protein. RESULTS: RKIP expression was high in breast duct epithelia and retained to varying degrees in primary breast tumors. However, in lymph node metastases, RKIP expression was highly significantly reduced or lost (P = 0.000003). No significant correlations were observed between RKIP expression and histologic type, tumor differentiation grade, size, or estrogen receptor status. CONCLUSION: This is the first study of RKIP expression in a large clinical cohort. It confirms the results of cell culture and animal studies, suggesting that in human breast cancer, RKIP is a metastasis suppressor gene whose expression must be down-regulated for metastases to develop. RKIP expression is independent of other markers for breast cancer progression and prognosis.