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Tamoxifen induces apoptosis through cancerous inhibitor of protein phosphatase 2A-dependent phospho-Akt inactivation in estrogen receptor-negative human breast cancer cells.
ABSTRACT: 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.
Project description:We tested the efficacy of lapatinib, a dual tyrosine kinase inhibitor which interrupts the HER2 and epidermal growth factor receptor (EGFR) pathways, in a panel of triple-negative breast cancer (TNBC) cells, and examined the drug mechanism. Lapatinib showed an anti-proliferative effect in HCC 1937, MDA-MB-468, and MDA-MB-231 cell lines. Lapatinib induced significant apoptosis and inhibited CIP2A and p-Akt in a dose and time-dependent manner in the three TNBC cell lines. Overexpression of CIP2A reduced lapatinib-induced apoptosis in MDA-MB-468 cells. In addition, lapatinib increased PP2A activity (in relation to CIP2A inhibition). Moreover, lapatinib-induced apoptosis and p-Akt downregulation was attenuated by PP2A antagonist okadaic acid. Furthermore, lapatinib indirectly decreased CIP2A transcription by disturbing the binding of Elk1 to the CIP2A promoter. Importantly, lapatinib showed anti-tumor activity in mice bearing MDA-MB-468 xenograft tumors, and suppressed CIP2A as well as p-Akt in these xenografted tumors. In summary, inhibition of CIP2A determines the effects of lapatinib-induced apoptosis in TNBC cells. In addition to being a dual tyrosine kinase inhibitor of HER2 and EGFR, lapatinib also inhibits CIP2A/PP2A/p-Akt signaling in TNBC cells.
Project description:BACKGROUND:Triple-negative breast cancer (TNBC) remains difficult to be targeted. SET and cancerous inhibitor of protein phosphatase 2A (CIP2A) are intrinsic protein-interacting inhibitors of protein phosphatase 2A (PP2A) and frequently overexpressed in cancers, whereas reactivating PP2A activity has been postulated as an anti-cancer strategy. Here we explored this strategy in TNBC. METHODS:Data from The Cancer Genome Atlas (TCGA) database was analyzed. TNBC cell lines were used for in vitro studies. Cell viability was examined by MTT assay. The apoptotic cells were examined by flow cytometry and Western blot. A SET-PP2A protein-protein interaction antagonist TD19 was used to disrupt signal transduction. In vivo efficacy of TD19 was tested in MDA-MB-468-xenografted animal model. FINDINGS:TCGA data revealed upregulation of SET and CIP2A and positive correlation of these two gene expressions in TNBC tumors. Ectopic SET or CIP2A increased cell viability, migration, and invasion of TNBC cells. Notably ERK inhibition increased PP2A activity. ERK activation is known crucial for Elk-1 activity, a transcriptional factor regulating CIP2A expression, we hypothesized an oncogenic feedforward loop consisting of pERK/pElk-1/CIP2A/PP2A. This loop was validated by knockdown of PP2A and ectopic expression of Elk-1, showing reciprocal changes in loop members. In addition, ectopic expression of SET increased pAkt, pERK, pElk-1 and CIP2A expressions, suggesting a positive linkage between SET and CIP2A signaling. Moreover, TD19 disrupted this CIP2A-feedforward loop by restoring PP2A activity, demonstrating in vitro and in vivo anti-cancer activity. Mechanistically, TD19 downregulated CIP2A mRNA via inhibiting pERK-mediated Elk-1 nuclear translocation thereby decreased Elk-1 binding to the CIP2A promoter. INTERPRETATION:These findings suggested that a novel oncogenic CIP2A-feedforward loop contributes to TNBC progression and targeting SET to disrupt this oncogenic CIP2A loop showed therapeutic potential in TNBC.
Project description:Protein phosphatase 2A (PP2A) is a tumor suppressor, which is functionally defective in various cancers. Previously, we found that PP2A activity determined the anticancer effect of bortezomib and erlotinib in hepatocellular carcinoma (HCC) cells. Here, we tested a novel erlotinib derivative, TD52, in four HCC cell lines, PLC5, Huh-7, Hep3B and Sk-Hep1. Using MTT and flow cytometry, we showed that TD52 had more potent apoptotic effects than erlotinib in HCC cells. TD52-induced apoptosis was associated with dose- and time- dependent reactivation of PP2A and downregulation of cancerous inhibitor of protein phosphatase 2A (CIP2A) and p-Akt. Inhibition of PP2A or ectopic expression of CIP2A or Akt in PLC5 cells abolished the effects of TD52. Furthermore, we demonstrated that TD52 affected the binding of Elk-1 to the proximal promoter of the CIP2A gene, thus downregulating transcription of CIP2A. Importantly, TD52-induced tumor inhibition was associated with reactivation of PP2A and downregulation of CIP2A and p-Akt in vivo. In conclusion, we found that enhancement of PP2A activity by inhibition of CIP2A determines the apoptotic effect induced by TD52. Our findings disclose the therapeutic mechanism of this novel targeted agent, and suggest the therapeutic potential and feasibility of developing PP2A enhancers as a novel anticancer strategy.
Project description:Nearly 40 000 women die annually from breast cancer in the United States. Clinically available targeted breast cancer therapy is largely ineffective in triple negative breast cancer (TNBC), characterized by tumors that lack expression of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (Her2). TNBC is associated with a poor prognosis. Previous reports show that aryl hydrocarbon receptor (AhR) partial agonist 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203) selectively inhibits the growth of breast cancer cells, including those of the TNBC subtype. We previously demonstrated that 5F 203 induced the expression of putative tumor suppressor gene cytoglobin (CYGB) in breast cancer cells. In the current study, we determined that 5F 203 induces apoptosis and caspase-3 activation in MDA-MB-468 TNBC cells and in T47D ER+ PR + Her2 - breast cancer cells. We also show that caspases and CYGB promote 5F 203-mediated apoptosis in MDA-MB-468 cells. 5F 203 induced lysosomal membrane permeabilization (LMP) and cathepsin B release in MDA-MB-468 and T47D cells. In addition, silencing CYGB attenuated the ability of 5F 203 to induce caspase-3/-7 activation, proapoptotic gene expression, LMP, and cathepsin B release in MDA-MB-468 cells. Moreover, 5F 203 induced CYGB protein expression, proapoptotic protein expression, and caspase-3 cleavage in MDA-MB-468 cells and in MDA-MB-468 xenograft tumors grown orthotopically in athymic mice. These data provide a basis for the development of AhR ligands with the potential to restore CYGB expression as a novel strategy to treat TNBC.
Project description:One of the factors that has increased the incidence and worse prognosis of breast cancer is obesity. In this condition, high amounts of leptin are secreted, which have proliferative, mitogenic, antiapoptotic, and proinflammatory activity that may be antagonistic to treatment with tamoxifen, considered the first choice. The modulation evaluation of leptin receptor expression in the presence of leptin and tamoxifen stimuli was performed in breast cancer cell lines MCF 7, MDA MB 231 and HCC 1937 as a model of initial approach for the study of breast cancer subtypes and their behavior to the action response of adipokines and their possible relationship with the mechanism of resistance to chemotherapeutics such as tamoxifen in ER positive cell lines and triple negative marker. It was determined that leptin increases the proliferation of the three breast cancer cell lines and tamoxifen is able to exert an antiproliferative effect on them, however, it was identified that the ability of tamoxifen to decrease the proliferation of cancer cells is diminished in the presence of leptin, in addition to changes in the modulation of the expression of its receptor. It was determined that tamoxifen induces a greater modulation of the expression of ObRb in cell lines, which may be related to the decrease of its antiproliferative activity, while leptin generates a proliferative effect in the three cell lines and could participate in the tamoxifen treatment resistance mechanism.
Project description:We have previously reported arginase expression in human breast cancer cells and demonstrated that the inhibition of arginase by N(?) hydroxy L-arginine (NOHA) in MDA-MB-468 cells induces apoptosis. However, arginase expression and its possible molecular targets in human breast tumor samples and potential clinical implications have not been fully elucidated. Here, we demonstrate arginase expression in human breast tumor samples, and several established breast cancer cell lines, in which NOHA treatment selectively inhibits cell proliferation. The over-expression of Bcl2 in MDA-MB-468 cells abolished NOHA-induced apoptosis, suggesting that the mitochondria may be the main site of NOHA's action. We, therefore, undertook a proteomics approach to identify key mitochondrial targets of arginase in MDA-MB-468 cells. We identified 54 non-mitochondrial and 13 mitochondrial proteins that were differentially expressed in control and NOHA treated groups. Mitochondrial serine hydroxymethyltransferase (mSHMT) was identified as one of the most promising targets of arginase. Both arginase II (Arg II) and mSHMT expressions were higher in human breast tumor tissues compared to the matched normal and there was a strong correlation between Arg II and mSHMT protein expression. MDA-MB-468 xenografts had significant upregulation of Arg II expression that preceded the induction of mSHMT expression. Small inhibitory RNA (siRNA)-mediated inhibition of Arg II in MDA-MB-468 and HCC-1806 cells led to significant inhibition of both the mSHMT gene and protein expression. As mSHMT is a key player in folate metabolism, our data provides a novel link between arginine and folate metabolism in human breast cancer, both of which are critical for tumor cell proliferation.
Project description:Numerous studies have implicated the aryl hydrocarbon receptor (AhR) as a potential therapeutic target for several human diseases, including estrogen receptor alpha (ER?) positive breast cancer. Aminoflavone (AF), an activator of AhR signaling, is currently undergoing clinical evaluation for the treatment of solid tumors. Of particular interest is the potential treatment of triple negative breast cancers (TNBC), which are typically more aggressive and characterized by poorer outcomes. Here, we examined AF's effects on two TNBC cell lines and the role of AhR signaling in AF sensitivity in these model cell lines.AF sensitivity in MDA-MB-468 and Cal51 was examined using cell counting assays to determine growth inhibition (GI50) values. Luciferase assays and qPCR of AhR target genes cytochrome P450 (CYP) 1A1 and 1B1 were used to confirm AF-mediated AhR signaling. The requirement of endogenous levels of AhR and AhR signaling for AF sensitivity was examined in MDA-MB-468 and Cal51 cells stably harboring inducible shRNA for AhR. The mechanism of AF-mediated growth inhibition was explored using flow cytometry for markers of DNA damage and apoptosis, cell cycle analysis, and ?-galactosidase staining for senescence. Luciferase data was analyzed using Student's T test. Three-parameter nonlinear regression was performed for cell counting assays.Here, we report that ER?-negative TNBC cell lines MDA-MB-468 and Cal51 are sensitive to AF. Further, we presented evidence suggesting that neither endogenous AhR expression levels nor downstream induction of AhR target genes CYP1A1 and CYP1B1 is required for AF-mediated growth inhibition in these cells. Between these two ER? negative cell lines, we showed that the mechanism of AF action differs slightly. Low dose AF mediated DNA damage, S-phase arrest and apoptosis in MDA-MB-468 cells, while it resulted in DNA damage, S-phase arrest and cellular senescence in Cal51 cells.Overall, this work provides evidence against the simplified view of AF sensitivity, and suggests that AF could mediate growth inhibitory effects in ER?-positive and negative breast cancer cells, as well as cells with impaired AhR expression and signaling. While AF could have therapeutic effects on broader subtypes of breast cancer, the mechanism of cytotoxicity is complex, and likely, cell line- and tumor-specific.
Project description:Triple-negative breast cancer (TNBC) has the poorest prognosis among all types of breast cancer and there is yet no effective therapy. Chemotherapy is the traditional standard of care for patients with TNBC; however, treatment of TNBC with chemotherapy may lead to the enrichment of cancer stem cells (CSCs), which exhibitan enhanced capacity for self-renewal, tumor initiation and metastasis. The present study demonstrated that bufalin, a small molecular compound used in traditional Chinese medicine, exerted anticancer effects on a wide range of cancer cell lines, inhibited cell proliferation through inducing G2/M cell cycle arrest, and triggered apoptosis in the TNBC cell lines MDA-MB-231 and HCC-1937. Consistently, bufalin markedly suppressed TNBC growth in a cell line-derived xenograft model. More importantly, unlike common chemotherapeutic drugs, bufalin reduced the stemness of TNBC stem cells. A mechanistic study suggested that bufalin may suppress the proliferation of TNBC stem cells by inhibiting the expression of octamer-binding transcription factor 4 (OCT4) and sex determining region Y-box 2 (SOX2) in MDA-MB-231 and HCC-1937 cells. These results indicated that bufalin may hold promise as a therapeutic agent in TNBC, and its effects may be mediated through the SOX2/OCT4 axis.
Project description:BACKGROUND:Semaphorin 4D (Sema4D) is highly expressed in certain types of tumors and functions in the regulation of tumor angiogenesis and growth. However, it is still not clear regarding the roles of Sema4D in breast cancer. This study was designed to explore the effects of Sema4D on proliferation, cell cycle progression, apoptosis, invasion, migration, tumor growth, and angiogenesis in breast cancer. MATERIALS AND METHODS:The expression level of Sema4D was investigated in MCF10A, 184A1, HCC1937, MDA-MB-468, MDA-MB-231, Hs578T, BT474, MCF-7, and T47D breast cancer cell lines by Western blotting analysis. Sema4D downregulation or overexpression was established by infection with lentiviruses-encoding Sema4D short hairpin RNA (shRNA) or Sema4D. To evaluate the effects of Sema4D on cell proliferation, cell cycle progression, apoptosis, invasion, and migration of MDA-MB-231 and MDA-MB-468 cells, methods including MTT assay, flow cytometry, wound healing assay, and transwell experiments were applied. BALB/c nude mice were injected with MDA-MB-231 cells, which were respectively infected with lentiviruses-encoding Sema4D, Sema4D shRNA, and GFP, followed by tumor angiogenesis assay. RESULTS:Sema4D was expressed at higher levels in breast cancer cell lines compared with the normal human breast epithelial cell lines, especially in MDA-MB-231 and MDA-MB-468 cells. Cell proliferation ability was remarkably inhibited in Sema4D downregulated condition, whereas the proportions of cells in the G0/G1 phase and apoptosis increased in MDA-MB-231 and MDA-MB-468 cells. In addition, the invasion and migration abilities of these cells were obviously reduced. Xenograft growth as well as angiogenesis was inhibited when infected with lentiviruses-encoding Sema4D shRNA in vivo. CONCLUSION:Downregulation of Sema4D had notable influence on cell proliferation ability, invasion, migration, and apoptosis of both MDA-MB-231 and MDA-MB-468 cells. Furthermore, infection with lentiviruses-encoding Sema4D shRNA obviously inhibited tumor growth and angiogenesis in BALB/c nude mice. Our results showed that Sema4D may represent a novel therapeutic target for human breast cancer.