Protein kinase Cα suppresses the expression of STC1 in MDA-MB-231 breast cancer cells

Several protein kinase C (PKC) isoforms have been shown to influence different cellular processes that may contribute to the malignancy of breast cancer cells. To obtain insight into mechanisms mediating the PKC effects, global gene expression was analyzed in MDA-MB-231 breast cancer cells in which PKCα, PKCδ or PKCε had been down-regulated with siRNA. Gene set enrichment analyses revealed that hypoxia-induced genes were enriched among genes that increased in PKCα-down-regulated cells. The STC1 mRNA, encoding stanniocalcin 1, was particularly up-regulated following depletion of PKCα and was also induced by hypoxia. Both hypoxia and PKCα down-regulation also led to increased STC1 protein levels. The results demonstrate that PKCα suppresses the expression of STC1 in breast cancer cells.     

Protein kinase C alpha expression in breast and ovarian cancer

In recent years research has focused on the development of specific, targeted drugs to treat cancer. One approach has been to block intracellular signaling proteins, such as protein kinase C alpha (PKC-alpha). To help support the rationale for clinical studies of a PKC-alpha-targeted therapy in breast and ovarian cancers, we reviewed publications studying PKC-alpha expression in these tumors. Since these investigations were mostly performed in cell lines, we supplemented this review with some preliminary findings from studies examining PKC-alpha expression in tumor tissue biopsies obtained from patients with breast and ovarian cancer. Based on the reviewed publications using representative cell lines and our preliminary findings on tumor tissue of patients with breast cancer, we infer that PKC-alpha levels may especially be increased in breast cancer patients with low or negative estrogen receptor (ER) levels. Thus, clinical studies determining efficacy of selective or specific inhibitors of PKC-alpha should include determination of ER status in order to help answer whether blocking PKC-alpha in patients with low or absent ER can result in clinical benefit.     

Protein kinase C zeta is required for epidermal growth factor-induced chemotaxis of human breast cancer cells

Chemotaxis plays an important role in cancer cell metastasis. In this study, we showed that epidermal growth factor (EGF) was a more potent chemoattractant than chemokine SDF-1alpha/CXCL12 for human breast cancer cell MDA-MB-231. Different inhibitors were used to evaluate the involvement of 12 protein kinase C (PKC) isotypes in the chemotactic signaling pathway. Chelerythrine chloride, an inhibitor of all PKC isotypes, blocked chemotaxis, whereas inhibitors of classic and novel PKC, such as G?6976, G?6850, or calphostin C, only impaired EGF-induced chemotaxis to a minor extent by not greater-than32% inhibition. These data suggested that atypical PKC were involved. The ligand-induced actin polymerization and cell adhesion were also similarly dependent on atypical PKC. Immunofluorescent staining showed an EGF-induced, LY294002-sensitive translocation of PKCzeta from the cytosol to the plasma membrane, indicating that EGF was capable of activating PKCzeta, probably via phosphoinositide 3 kinases. A myristoylated PKCzeta pseudosubstrate blocked the chemotaxis with an IC(50) of 20 mumol/L. To expand our investigation, we further showed that in MCF-7 and T47D, two additional human breast cancer cell lines, EGF-activated PKCzeta and the PKCzeta pseudosubstrate, inhibited chemotaxis. Taken together, our data suggest that PKCzeta is an essential component of the EGF-stimulated chemotactic signaling pathway in human breast cancer cells.     

Antiestrogen regulation of cell cycle progression and cyclin D1 gene expression in MCF-7 human breast cancer cells

The molecular mechanisms by which antiestrogens inhibit breast cancer cell proliferation are not well understood. Using cultured breast cancer cell lines, we studied the effects of antiestrogens on proliferation and cell cycle progression and used this information to select candidate cell cycle regulatory genes that are potential targets for antiestrogens. Under estrogen- and serum-free conditions antiestrogens inhibited proliferation of MCF-7 cells stimulated with insulin. Cells were blocked at a point in G₁ phase. These effects are comparable with those in serum- and estrogen-containing medium and were also seen to a lesser degree in nude mice bearing MCF-7 tumors. Similar observations with other peptide mitogens suggest that the process inhibited by antiestrogens is common to estrogen and growth factor activated pathways. Other studies have identified G₁ cyclins as potential targets for growth factor and steroid hormone/steroid antagonist regulation of breast epithelial cell proliferation. In MCF-7 cells growing in the presence of fetal calf serum, cyclin D1 mRNA was rapidly down-regulated by steroidal and nonsteroidal antiestrogens by an apparently estrogen receptor mediated mechanism. Cyclin D1 gene expression was maximally inhibited before effects on entry into S phase and inhibition was therefore not merely a consequence of changes in cell cycle progression. Together with data on the effects of antiestrogens in serum-free conditions [1], these results suggest down-regulation of cyclin D1 by antiestrogens may be a general phenomenon in estrogen receptor-positive breast cancer cells, independent of culture conditions and class of antiestrogen. These observations are compatible with the hypothesis that reductions in cyclin D1 levels may mediate in part the action of antiestrogens in blocking entry of cells into S phase.     

乳腺癌表达细胞周期蛋白依赖性激酶抑制物p27kip1的预后意义

研究ｐ２７＾ｋｉｐｌ在乳腺癌组织中的表达水平,及其与生物学行为的关系和对预后的作用。方法以免疫组化法检测１８１例乳腺癌组织ｐ２７＾ｋｉｐｌ蛋白表达水平,其中９１例进行了凋亡指数、凋亡相关蛋白ｂｃｌ－２、ｂａｘ的测定,另有１０６例检测了ｐ２１＾ＷＡＦ１／ＣＩＰＩ、ｐ５３、ｍｄｍ２蛋白表达水平。结果本组１８１例中,ｐ２７＾ｋｉｐｌ蛋白阳性１２５例,占６９．１％;低表达者１２１例,占６６．９％;高表达者     

Protein kinase D1 regulates matrix metalloproteinase expression and inhibits breast cancer cell invasion

Introduction  

The biological and molecular events that regulate the invasiveness of breast tumour cells need to be further revealed to develop effective therapies that stop breast cancer from expanding and metastasising.     

Protein kinase D1 regulates matrix metalloproteinase expression and inhibits breast cancer cell invasion

Introduction

Unclassified variants (UVs) in the BRCA1/BRCA2 genes are a frequent problem in counseling breast cancer and/or ovarian cancer families. Information about cancer family history is usually available, but has rarely been used to evaluate UVs. The aim of the present study was to identify which is the best combination of clinical parameters that can predict whether a UV is deleterious, to be used for the classification of UVs.

Methods

We developed logistic regression models with the best combination of clinical features that distinguished a positive control of BRCA pathogenic variants (115 families) from a negative control population of BRCA variants initially classified as UVs and later considered neutral (38 families).

Results

The models included a combination of BRCAPRO scores, Myriad scores, number of ovarian cancers in the family, the age at diagnosis, and the number of persons with ovarian tumors and/or breast tumors. The areas under the receiver operating characteristic curves were respectively 0.935 and 0.836 for the BRCA1 and BRCA2 models. For each model, the minimum receiver operating characteristic distance (respectively 90% and 78% specificity for BRCA1 and BRCA2) was chosen as the cutoff value to predict which UVs are deleterious from a study population of 12 UVs, present in 59 Dutch families. The p.S1655F, p.R1699W, and p.R1699Q variants in BRCA1 and the p.Y2660D, p.R2784Q, and p.R3052W variants in BRCA2 are classified as deleterious according to our models. The predictions of the p.L246V variant in BRCA1 and of the p.Y42C, p.E462G, p.R2888C, and p.R3052Q variants in BRCA2 are in agreement with published information of them being neutral. The p.R2784W variant in BRCA2 remains uncertain.

Conclusions

The present study shows that these developed models are useful to classify UVs in clinical genetic practice.     

细胞周期蛋白D1表达与乳腺癌新辅助化疗关系的研究

目的 观察乳腺癌新辅助化疗(NAC)前后cyclin D1表达情况,探讨其与化疗效果之间的关系.方法 选择84例乳腺癌患者经空心针穿刺获取组织学样本并加以验证,经3～4个周期ET方案[吡柔比星(THP)+多西紫杉醇(TXT)]NAC,采用免疫组织化学Envision二步法检测化疗前、后cyclinD1的表达变化情况.结果 84例患者中完全缓解(CR)4例(4.76％)(其中病理CR 2例),部分缓解(PR) 54例(64.29％),稳定(SD) 26例(30.95％),无疾病进展(PD)患者.NAC后乳腺癌组织中cyclin D1表达降低[65.48％(55/84)],与化疗前[39.29％(33/84)]相比,差异有统计意义(x 2=11.55,P=0.001).NAC后cyclin D1阳性表达转为阴性表达者临床缓解率[86.36％(19/22)]高于未转为阴性表达者[45.45％(15/33)],差异有统计学意义(x2=9.359,P=0.002).结论 NAC降低乳腺癌组织中的cyclinD1表达,且NAC后cyclinD1表达转阴者化疗效果提高,可以评估NAC的有效性.     

Targeting GLI1 expression in human inflammatory breast cancer cells enhances apoptosis and attenuates migration

Background:

Inflammatory breast cancer (IBC) is an aggressive subtype of breast cancer with distinct molecular profiles. Gene expression profiling previously identified sonic hedgehog (SHH) as part of a gene signature that is differentially regulated in IBC patients.

Methods:

The effects of reducing GLI1 levels on protein expression, cell proliferation, apoptosis and migration were determined by immunoblots, MTT assay, Annexin-V/PI assay and conventional and automated cell migration assays.

Results:

Evaluation of a panel of breast cancer cell lines revealed elevated GLI1 expression, typically a marker for hedgehog-pathway activation, in a triple-negative, highly invasive IBC cell line, SUM149 and its isogenic-derived counterpart rSUM149 that has acquired resistance to ErbB1/2 targeting strategies. Downregulation of GLI1 expression in SUM149 and rSUM149 by small interfering RNA or a small molecule GLI1 inhibitor resulted in decreased proliferation and increased apoptosis. Further, GLI1 suppression in these cell lines significantly inhibited cell migration as assessed by a wound-healing assay compared with MCF-7, a non-invasive cell line with low GLI1 expression. A novel high-content migration assay allowed us to quantify multiple effects of GLI1 silencing including significant decreases in cell distance travelled and linearity of movement.

Conclusion:

Our data reveal a role for GLI1 in IBC cell proliferation, survival and migration, which supports the feasibility of targeting GLI1 as a novel therapeutic strategy for IBC patients.     

蛋白激酶C-βI在乳腺癌组织中的表达及意义

目的观察蛋白激酶C(proteinkinaseC,PKC)同工酶在乳腺癌组织中的表达状况,探讨癌细胞信息传递的分子生物学机制。方法应用半定量RT-PCR检测了31例乳腺癌组织与12例癌旁正常乳腺组织中PKC-βΙmRNA表达;应用Westernblot检测以上组织中PKC-βΙ蛋白表达。结果乳腺癌组织中PKC-βΙmRNA与蛋白表达均高于癌旁正常乳腺组织(P<0.001)。结论乳腺癌组织中存在PKC-βΙ同工酶过度表达,它可能介导乳腺癌细胞分化的信号转导过程。