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PLD1 is overexpressed in an ER-negative MCF-7 cell line variant and a subset of phospho-Akt-negative breast carcinomas.

ABSTRACT: We have used a novel variant of the human oestrogen receptor (ER)-positive MCF-7 cell line, TMX2-28, as a model to study breast cancer. TMX2-28 cells show no detectable levels of mRNA or protein expression for the ER and express basal cytokeratins (CKs) 5, 14, and 17. cDNA microarray comparison between TMX2-28 and its parent cell line, MCF-7, identified 1402 differentially expressed transcripts, one of which was, phospholipase D1 (PLD1). Using real-time RT-PCR, we confirmed that PLD1 mRNA levels are 10-fold higher in TMX2-28 cells than in MCF-7 cells. We next examined PLD1 expression in human breast carcinomas. Phospholipase D1 mRNA levels were higher in breast tumours that expressed high-mRNA levels of basal CKs 5 and/or 17, but PLD1 mRNA levels were not significantly higher in ER-negative tumours. Phospholipase D1 protein was overexpressed in 10 of 42 (24%) breast tumours examined by IHC. Phospholipase D1 was overexpressed in 6 of 31 ER-positive tumours and 4 of 11 ER-negative tumours. Phospholipase D1 was overexpressed in three of the four tumours that showed high CK5/17 expression. Five PLD1-positive tumours were negative for phospho-Akt expression, but positive for phospho-mammalian target of rapamycin (mTOR) expression. The other five PLD1-positive breast tumours showed positive expression for phospho-Akt; however, only two of these cases were positive for phospho-mTOR. In this study, we report that PLD1 and phospho-mTOR are coexpressed in a subset of phospho-Akt-negative breast carcinomas.

SUBMITTER: Gozgit JM

PROVIDER: S-EPMC2360386 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundHepatocellular carcinoma (HCC) is the fourth fatal malignant tumour type worldwide. However, the exact molecular mechanism involved in HCC progression remains unclear.MethodsThree pairs of HCC and matched portal vein tumour thrombus (PVTT) tissue samples were analysed by isobaric tags for relative and absolute quantification (iTRAQ) assay to investigate the differentially expressed proteins. Real-time quantitative PCR, immunostaining, and immunoblotting were performed to detect cofilin 1 (CFL1) in HCC and non-tumour tissues. CCK8 and EdU, and Transwell assays, respectively, determined cell proliferation, migration, and invasion of HCC cells. Further, subcutaneous and tail vein injection were performed in nude mice for investigating HCC growth and lung metastasis in vivo. Regulatory effect of hypoxia-inducible factor-1α (HIF-1α) on CFL1 was confirmed by chromatin immunoprecipitation (ChIP) assay. Finally, interaction between CFL1 and phospholipase D1 (PLD1) was studied using immunoprecipitation (IP) assay.ResultsThe iTRAQ analysis identified expression of CFL1 to be significantly upregulated in PVTT than in HCC tissues. Increased expression of CFL1 was closely associated with unfavourable clinical features, and was an independent risk predictor of overall survival in HCC patients. The knockdown of CFL1 inhibited cell growth viability, invasiveness, and epithelial-mesenchymal transformation (EMT) in HCC cells. Furthermore, CFL1 silencing significantly suppressed the growth and lung metastasis of HCC cells in nude mice. Next, HIF-1α directly regulated CFL1 transcription by binding to the hypoxia-responsive element (HRE) in the promoter. Moreover, we disclosed the interaction between CFL1 and PLD1 in HCC cells using IP assay. Mechanistically, CFL1 maintained PLD1 expression by repressing ubiquitin-mediated protein degradation, thereby activating AKT signalling in HCC cells. Notably, the CFL1/PLD1 axis was found mediating the hypoxia-induced activation of the AKT pathway and EMT.ConclusionThe analysis suggests that hypoxia-induced CFL1 increases the proliferation, migration, invasion, and EMT in HCC by activating the PLD1/AKT pathway.

Project description:BackgroundThe origin and the contribution of breast tumor heterogeneity to its progression are not clear. We investigated the effect of a growing orthotopic tumor formed by an aggressive estrogen receptor (ER)-negative breast cancer cell line on the metastatic potential of a less aggressive ER-positive breast cancer cell line for the elucidation of how the presence of heterogeneous cancer cells might affect each other's metastatic behavior.MethodsER positive ZR-75-1/GFP/puro cells, resistant to puromycin and non-tumorigenic/non-metastatic without exogenous estrogen supplementation, were injected intracardiacally into mice bearing growing orthotopic tumors, formed by ER negative MDA-MB-231/GFP/Neo cells resistant to G418. A variant cell line B6, containing both estrogen-dependent and -independent cells, were isolated from GFP expressing cells in the bone marrow and re-inoculated in nude mice to generate an estrogen-independent cell line B6TC.ResultsThe presence of ER negative orthotopic tumors resulted in bone metastasis of ZR-75-1 without estrogen supplementation. The newly established B6TC cell line was tumorigenic without estrogen supplementation and resistant to both puromycin and G418 suggesting its origin from the fusion of MDA-MB-231/GFP/Neo and ZR-75-1/GFP/puro in the mouse bone marrow. Compared to parental cells, B6TC cells were more metastatic to lung and bone after intracardiac inoculation. More significantly, B6TC mice also developed brain metastasis, which was not observed in the MDA-MB-231/GFP/Neo cell-inoculated mice. Low expression of ERα and CD24, and high expression of EMT-related markers such as Vimentin, CXCR4, and Integrin-β1 along with high CD44 and ALDH expression indicated stem cell-like characteristics of B6TC. Gene microarray analysis demonstrated a significantly different gene expression profile of B6TC in comparison to those of parental cell lines.ConclusionsSpontaneous generation of the novel hybrid cell line B6TC, in a metastatic site with stem cell-like properties and propensity to metastasize to brain, suggest that cell fusion can contribute to tumor heterogeneity.

Project description:INTRODUCTION:Tamoxifen, a selective estrogen receptor (ER) modulator, may affect cancer cell survival through mechanisms other than ER antagonism. In the present study, we tested the efficacy of tamoxifen in a panel of ER-negative breast cancer cell lines and examined the drug mechanism. METHODS:In total, five ER-negative breast cancer cell lines (HCC-1937, MDA-MB-231, MDA-MB-468, MDA-MB-453 and SK-BR-3) were used for in vitro studies. Cellular apoptosis was examined by flow cytometry and Western blot analysis. Signal transduction pathways in cells were assessed by Western blot analysis. The in vivo efficacy of tamoxifen was tested in xenograft nude mice. RESULTS:Tamoxifen induced significant apoptosis in MDA-MB-231, MDA-MB-468, MDA-MB-453 and SK-BR-3 cells, but not in HCC-1937 cells. Tamoxifen-induced apoptosis was associated with inhibition of cancerous inhibitor of protein phosphatase 2A (CIP2A) and phospho-Akt (p-Akt) in a dose-dependent manner. Ectopic expression of either CIP2A or Akt protected MDA-MB-231 cells from tamoxifen-induced apoptosis. In addition, tamoxifen increased protein phosphatase 2A (PP2A) activity, and tamoxifen-induced apoptosis was attenuated by the PP2A antagonist okadaic acid in the sensitive cell lines, but not in resistant HCC-1937 cells. Moreover, silencing CIP2A by small interfering RNA sensitized HCC-1937 cells to tamoxifen-induced apoptosis. Furthermore, tamoxifen regulated CIP2A protein expression by downregulating CIP2A mRNA. Importantly, tamoxifen inhibited the in vivo growth of MDA-MB-468 xenograft tumors in association with CIP2A downregulation, whereas tamoxifen had no significant effect on CIP2A expression and anti-tumor growth in HCC-1937 tumors. CONCLUSIONS:Inhibition of CIP2A determines the effects of tamoxifen-induced apoptosis in ER-negative breast cancer cells. Our data suggest a novel "off-target" mechanism of tamoxifen and suggest that CIP2A/PP2A/p-Akt signaling may be a feasible anti-cancer pathway.

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PLD1 is overexpressed in an ER-negative MCF-7 cell line variant and a subset of phospho-Akt-negative breast carcinomas.

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