34 research outputs found
Abemaciclib in Combination With Endocrine Therapy for Patients With Hormone Receptor-Positive, HER2-Negative Metastatic Breast Cancer: A Phase 1b Study
Background Cyclin-dependent kinases (CDK) 4 and 6 regulate G1 to S cell cycle progression and are often altered in cancers. Abemaciclib is a selective inhibitor of CDK4 and CDK6 approved for administration on a continuous dosing schedule as monotherapy or as combination therapy with an aromatase inhibitor or fulvestrant in patients with advanced or metastatic breast cancer. This Phase 1b study evaluated the safety and tolerability, pharmacokinetics, and antitumor activity of abemaciclib in combination with endocrine therapy for metastatic breast cancer (MBC), including aromatase inhibitors (letrozole, anastrozole, or exemestane) or tamoxifen. Patients and Methods Women β₯18 years old with hormone receptor positive (HR+), human epidermal growth factor receptor 2 negative (HER2-) MBC were eligible for enrollment. Eligibility included measurable disease or non-measurable but evaluable bone disease by Response Evaluation Criteria in Solid Tumours (RECIST) v1.1, Eastern Cooperative Oncology Group performance status 0β1, and no prior chemotherapy for metastatic disease. Adverse events were graded by the National Cancer Institute Common Terminology Criteria for Adverse Events v4.0 and tumor response were assessed by RECIST v1.1. Results Sixty-seven patients were enrolled and received abemaciclib 200 mg every 12 hours in combination with letrozole (Part A, n=20), anastrozole (Part B, n=16), tamoxifen (Part C, n=16), or exemestane (Part D, n=15). The most common treatment-emergent adverse events (TEAE) were diarrhea, fatigue, nausea, and abdominal pain. Grade 4 TEAEs were reported in five patients (one each with hyperglycemia, hypertension, neutropenia, procedural hemorrhage, and sepsis). There was no effect of abemaciclib or endocrine therapy on the pharmacokinetics of any combination study drug. Across all treated patients, the median progression-free survival was 25.4 months (95% confidence interval: 18.0, 35.8). The objective response rate was 38.9% in 36 patients with measurable disease. Conclusions Abemaciclib in combination with multiple endocrine therapy options exhibited manageable safety and promising antitumor activity in patients with HR+, HER2- MBC. Clinical Trial Registration https://clinicaltrials.gov/, identifier NCT0205713
Identification and Characterization of Nucleolin as a COUP-TFII Coactivator of Retinoic Acid Receptor Ξ² Transcription in Breast Cancer Cells
The orphan nuclear receptor COUP-TFII plays an undefined role in breast cancer. Previously we reported lower COUP-TFII expression in tamoxifen/endocrine-resistant versus sensitive breast cancer cell lines. The identification of COUP-TFII-interacting proteins will help to elucidate its mechanism of action as a transcriptional regulator in breast cancer.FLAG-affinity purification and multidimensional protein identification technology (MudPIT) identified nucleolin among the proteins interacting with COUP-TFII in MCF-7 tamoxifen-sensitive breast cancer cells. Interaction of COUP-TFII and nucleolin was confirmed by coimmunoprecipitation of endogenous proteins in MCF-7 and T47D breast cancer cells. In vitro studies revealed that COUP-TFII interacts with the C-terminal arginine-glycine repeat (RGG) domain of nucleolin. Functional interaction between COUP-TFII and nucleolin was indicated by studies showing that siRNA knockdown of nucleolin and an oligonucleotide aptamer that targets nucleolin, AS1411, inhibited endogenous COUP-TFII-stimulated RARB2 expression in MCF-7 and T47D cells. Chromatin immunoprecipitation revealed COUP-TFII occupancy of the RARB2 promoter was increased by all-trans retinoic acid (atRA). RARΞ²2 regulated gene RRIG1 was increased by atRA and COUP-TFII transfection and inhibited by siCOUP-TFII. Immunohistochemical staining of breast tumor microarrays showed nuclear COUP-TFII and nucleolin staining was correlated in invasive ductal carcinomas. COUP-TFII staining correlated with ERΞ±, SRC-1, AIB1, Pea3, MMP2, and phospho-Src and was reduced with increased tumor grade.Our data indicate that nucleolin plays a coregulatory role in transcriptional regulation of the tumor suppressor RARB2 by COUP-TFII
Targeting CDK4 and 6 in Cancer Therapy: Emerging Preclinical Insights Related to Abemaciclib
Pharmacologic inhibitors of cyclin-dependent kinases 4 and 6 (CDK4 and 6) are approved for the treatment of subsets of patients with hormone receptor positive (HR+) breast cancer (BC). In metastatic disease, strategies involving endocrine therapy combined with CDK4 and 6 inhibitors (CDK4 and 6i) improve clinical outcomes in HR+ BCs. CDK4 and 6i prevent retinoblastoma tumor suppressor protein phosphorylation, thereby blocking the transcription of E2F target genes, which in turn inhibits both mitogen and estrogen-mediated cell proliferation. In this review, we summarize preclinical data pertaining to the use of CDK4 and 6i in BC, with a particular focus on several of the unique chemical, pharmacologic, and mechanistic properties of abemaciclib. As research efforts elucidate the novel mechanisms underlying abemaciclib activity, potential new applications are being identified. For example, preclinical studies have demonstrated abemaciclib can exert antitumor activity against multiple tumor types and can cross the blood-brain barrier. Abemaciclib has also demonstrated distinct activity as a monotherapeutic in the treatment of BC. Accordingly, we also discuss how a greater understanding of mechanisms related to CDK4 and 6 blockade highlight abemaciclib's unique in-class properties, and could pave new avenues for enhancing its therapeutic efficacy
Reduced Expression of miR-200 Family Members Contributes to Antiestrogen Resistance in LY2 Human Breast Cancer Cells
<div><p>Introduction</p><p>The role of miRNAs in acquired endocrine-resistant breast cancer is not fully understood. One hallmark of tumor progression is epithelial-to-mesenchymal transition (EMT), characterized by a loss of cell adhesion resulting from reduced E-cadherin and increased cell mobility. miR-200 family members regulate EMT by suppressing expression of transcriptional repressors ZEB1/2. Previously we reported that the expression of miR-200a, miR-200b, and miR-200c was lower in LY2 endocrine-resistant, mesenchymal breast cancer cells compared to parental, endocrine sensitive, epithelial MCF-7 breast cancer cells. Here we investigated the regulation of miR-200 family members and their role in endocrine-sensitivity in breast cancer cells.</p><p>Results</p><p>miR-200 family expression was progressively reduced in a breast cancer cell line model of advancing endocrine/tamoxifen (TAM) resistance. Concomitant with miR-200 decrease, there was an increase in ZEB1 mRNA expression. Overexpression of miR-200b or miR-200c in LY2 cells altered cell morphology to a more epithelial appearance and inhibited cell migration. Further, miR-200b and miR-200c overexpression sensitized LY2 cells to growth inhibition by estrogen receptor (ER) antagonists TAM and fulvestrant. Knockdown of ZEB1 in LY2 cells recapitulated the effect of miR-200b and miR-200c overexpression resulting in inhibition of LY2 cell proliferation by TAM and fulvestrant, but not the aromatase inhibitor exemestane. Demethylating agent 5-aza-2β²-deoxycytidine (5-aza-dC) in combination with histone deacetylase inhibitor trichostatin A (TSA) increased miR-200b and miR-200c in LY2 cells. Concomitant with the increase in miR-200b and miR-200c, ZEB1 expression was decreased and cells appeared more epithelial in morphology and were sensitized to TAM and fulvestrant inhibition. Likewise, knockdown of ZEB1 increased antiestrogen sensitivity of LY2 cells resulting in inhibition of cell proliferation.</p><p>Conclusions</p><p>Our data indicate that reduced miRNA-200b and miR-200c expression contributes to endocrine resistance in breast cancer cells and that the reduced expression of these miR-200 family members in endocrine-resistant cells can be reversed by 5-aza-dC+TSA.</p></div
Knockdown of miR-200b or miR-200c does not promote resistance of MCF-7 to 4-OHT or fulvestrant.
<p>MCF-7 cells were transfected with a negative control, anti-miR-200b, or anti-miR-200c. 1 d post transfection, cells were treated with 100 nM 4-OHT or 100 nM fulvestrant for 4 d prior to MTT assay. A, CT values for miR-200b and miR-200c in the cells transfected as indicated for 1 or 5 d. Values are the mean Β± SEM of 3 determinations. B, MTT values are the mean Β± SEM of 3 experiments. *p<0.05 versus EtOH-treated, control-transfected MCF-7 cells; <sup>#</sup>p<0.05 versus EtOH-treated, miR-200b- or miR-200c- transfected cells.</p
miR-200b and miR-200c inhibit LY2 cell migration in a wound healing assay.
<p>LY2 cells were plated in a 6-well plate (3000 cells/well), transfected with negative control, pre-miR-200b, or pre-miR-200c for 24 h. Cells were wounded by scratching using a p200 pipette tip at time zero (0 h). Cells were washed with medium to remove displaced cells. The wound closure was analyzed at the indicated times using NIH Image J software. Values are the mean Β± SEM of four separate measurements. *p<0.01 versus negative control-transfected cells. There were no statistical differences between cells transfected with pre-miR-200b versus pre-miR-200c.</p
Overexpression of miR-200b and miR-200c inhibits expression of mesenchymal markers and increases E-cadherin in LY2 cells.
<p>(AβC) LY2 cells were not transfected (Non-TF), Mock-transfected (RNAiMAX), or transfected with negative control, pre-miR-200b, or pre-miR-200c for 24 h before preparing RNA or WCE for subsequent analysis. A, <i>ZEB1</i>, <i>ZEB2</i>, E-cadherin (<i>CDH1</i>), and vimentin (<i>VIM</i>) expression was quantified by qPCR. Values are the mean Β± SEM. *p<0.05 versus negative control for each gene. BβC, Protein expression of Zeb1 as well as EMT markers N-cadherin, vimentin, and Slug, and epithelial marker E-cadherin was analyzed by western blotting normalized to the expression of Ξ±-tubulin. Non-transfected values are set to one.</p
5-aza-dC and TSA increase miR-200b and miR-200c expression in LY2 cells.
<p>(AβF) LY2 or MCF-7 cells were treated with 2.5 Β΅M 5-aza-dC for 72 h with 100 ng/ml TSA added for the last 16 h of treatment. The expression of miR-200b, miR-200c or ZEB1 mRNA was determined by qPCR. Values are the mean Β± SEM of triplicate determinations. G, LY2 cells were treated with 2.5 Β΅M 5-aza-dC (AZA) alone for 72 h or with 100 ng/ml TSA added for the last 24 h of treatment. Whole cell lysates were separated by 10% SDS-PAGE and western blotted for ZEB1 expression. The membrane was stripped and reprobed for Ξ±-tubulin. ZEB1/Ξ±-tubulin expression was normalized to non-treated control cells. Values are Β± SEM of duplicate determinations. *p<0.05 versus untreated.</p
ZEB1 mRNA expression in MCF-7, LCC1, LCC2, LCC9 and LY2 cells.
<p>Cells were serum-starved for 48 h and treated with EtOH (vehicle control), 10 nM E<sub>2</sub>, or 100 nM 4-OHT for 6 h. ZEB1 expression was quantified by qPCR. Values are the mean Β± SEM of 3 experiments and are expressed as fold relative to EtOH-treated MCF-7. *p<0.05 <i>versus</i> EtOH-treated MCF-7.</p
Model of function of miR-200 family members in endocrine resistance in breast cancer cells.
<p>A, In endocrine-sensitive breast cancer cells, <i>e.g</i>., MCF-7, miR-200b and miR-200c are expressed, resulting in low ZEB1 protein expression. The lack of ZEB1 allows expression of E-cadherin. Vimentin is not expressed. B, In endocrine-resistant cells that have undergone EMT, miR-200 family expression is low, resulting in increased ZEB1 protein which inhibits E-cadherin expression. Vimentin, N-cadherin, and Slug expression is increased. Slug directly inhibits miR-200b expression <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062334#pone.0062334-Liu1" target="_blank">[69]</a>. Grey lettering indicates reduced expression and dashed lines indicate reduced regulation.</p