Yingchun Zhao

let-7 microRNAs induce tamoxifen sensitivity by downregulation of estrogen receptor α signaling in breast cancer.

MicroRNAs (miRNAs) play an important regulatory role in breast tumorigenesis. Previously, we found that let-7 miRNAs were downregulated significantly in formalin-fixed paraffin-embedded (FFPE) breast cancer tissues. In this study, we further found that endogenous levels of let-7b and let-7i miRNAs are inversely correlated with levels of estrogen receptor (ER)-a36, a new variant of ER-α66, in the FFPE tissue set. Bioinformatic analysis suggested that ER-α36 may be another target of let-7 miRNAs. To test this hypothesis, cotransfection of let-7 mimics or inhibitors together with full-length or a fragment of ER-α36 3′UTR luciferase construct was performed, and we found that let-7b and let-7i mimics suppressed the activity of reporter gene significantly, which was enhanced remarkably by let-7b and let-7i inhibitors. Both mRNA and protein expression of ER-α36 were inhibited by let-7 mimics and enhanced by let-7 inhibitors. Furthermore, ER-α36 mediated nongenomic MAPK and Akt pathways were weakened by let-7b and let-7i mimics in triple negative breast cancer cell line MDA-MB-231. The reverse correlation between let-7 miRNAs and ER-α36 also exists in Tamoxifen (Tam)-resistant MCF7 cell line. Transfection of let-7 mimics to Tam-resistant MCF7 cells downregulated ER-α36 expression and enhanced the sensitivity of MCF7 cells to Tam in estrogen-free medium, which could be restored by overexpression of ER-α36 constructs without 3′UTR. Our results suggested a novel regulatory mechanism of let-7 miRNAs on ER-α36 mediated nongenomic estrogen signal pathways and Tam resistance.

Quantitative Proteomics and Biomarker Discovery in Human Cancer

“Early diagnosis and prevention is a key factor in reducing the mortality and morbidity of cancer. However, currently available screening tools lack enough sensitivity for early diagnosis. It is important to develop noninvasive techniques and methods that can screen and identify asymptomatic patients who have cancer. Biomarkers of cancer status can also serve as powerful tools in monitoring the course of cancer, and in determining the efficacy and safety of novel therapies. Thus, discovery of novel specific biomarkers are needed that may provide informative clues for early diagnosis and treatment of cancer. Recently, remarkable progress has been made in the development of new proteomics technology. The progress that has been made in this field is helpful in identifying biomarkers that can be used for early diagnosis of cancer and improving the understanding of the molecular etiological mechanism of cancer. This article describes the current state of the art in this field.”

Inhibition of protein phosphorylation in MIA pancreatic cancer cells: Confluence of metabolic and signaling pathways

Oxythiamine (OT), a transketolase inhibitor, is known to inhibit pancreatic cancer cell proliferation. In this study, we investigated the effect of inhibition of the transketolase pathway on signaling pathways in MIA PaCa cancer cells using in-house proteomic techniques. We hypothesized that OT alter protein phosphorylation thus affecting cell cycle arrest and cell proliferation. MIA PaCa-2 cells were cultured in media containing an algal (15)N amino acid mixture at 50% enrichment, with and without OT, to determine protein expression and synthesis. Analysis of cell lysates using two-dimensional gel electrophoresis matrix assisted laser desorption and ionization time-of-flight and time-of-flight mass spectrometry (2-DE-MALDI-TOF/TOF MS) identified 12 phosphor proteins that were significantly suppressed by OT treatment. Many of these proteins are involved in regulation of cycle activities and apoptosis. Among the proteins identified, expression of the phosphor heat shock protein 27 (Hsp27) was dramatically inhibited by OT treatment while the level of its total protein remained unchanged. Hsp27 expression and phosphorylation is known to be associated with drug resistance and cancer cell survival. The changes in phosphorylation of key proteins of cancer proliferation and survival suggest that protein phosphorylation is the confluence of the effects of OT on metabolic and signaling pathways.

Inhibition of glycogen phosphorylation induces changes in cellular proteome and signaling pathways in MIA pancreatic cancer cells

Objectives Novel quantitative proteomic approaches were used to study the effects of inhibition of glycogen phosphorylase on proteome and signaling pathways in MIA PaCa-2 pancreatic cancer cells. Methods We performed quantitative proteomic analysis in MIA PaCa-2 cancer cells treated with a stratified dose of CP-320626 (5-chloro-1H-indole-2-carboxylic acid [1-(4-fuorobenzyl)-2-(4-hydroxypiperidin-1-yl)-2 oxoethyl] amide) (25, 50, and 100 &mgr;M). The effect of metabolic inhibition on cellular protein turnover dynamics was also studied using the modified SILAC (stable isotope labeling with amino acids in cell culture) method. Results A total of 22 protein spots and 4 phosphoprotein spots were quantitatively analyzed. We found that dynamic expression of total proteins and phosphoproteins was significantly changed in MIA PaCa-2 cells treated with an incremental dose of CP-320626. Functional analyses suggested that most of the proteins differentially expressed were in the pathways of mitogen-activated protein kinase/extracellular signal–regulated kinase and tumor necrosis factor &agr;/nuclear factor &kgr;B. Conclusions Signaling pathways and metabolic pathways share many common cofactors and substrates forming an extended metabolic network. The restriction of substrate through 1 pathway such as inhibition of glycogen phosphorylation induces pervasive metabolomic and proteomic changes manifested in protein synthesis, breakdown, and posttranslational modification of signaling molecules. Our results suggest that quantitative proteomic is an important approach to understand the interaction between metabolism and signaling pathways. Abbreviations MALDI - matrix-assisted laser desorption ionization, TOF/TOF MS - time-of-flight/time-of-flight mass spectrometry, 2-DE - 2-dimensional electrophoresis, PMF - peptide mass fingerprinting, CP-320626 - 5-chloro-1H-indole-2-carboxylic acid [1-(4-fuorobenzyl)-2-(4-hydroxypiperidin-1-yl)-2 oxoethyl]amide

Smoke-Induced Signal Molecules in Bone Marrow Cells from Altered Low-Density Lipoprotein Receptor-Related Protein 5 Mice

Mechanism underlying smoke-induced loss of bone mass is unknown. In this study, we hypothesized that protein signals induced by smoking in bone marrow may be associated with the loss of bone mass. Using a proteomics approach, we identified 38 proteins differentially expressed in bone marrow cells from low-density lipoprotein receptor-related protein 5 (Lrp5) mice exposed to cigarette smoking. Smoking effects on protein expression in bone marrow among three genotypes (Lrp5(+/+), Lrp5(G171V), and Lrp5(-/-)) varied. On the basis of the ratio of protein expression induced by smoking versus nonsmoking, smoke induced protein expression significantly in wild-type mice compared to the other two genotypes (Lrp5(G171V) and Lrp5(-/-)). These proteins include inhibitors of β-catenin and proteins associated with differentiation of osteoclasts. We observed that S100A8 and S100A9 were overexpressed in human smokers compared to nonsmokers, which confirmed the effect of smoking on the expression of two proteins in Lrp5 mice, suggesting the role of these proteins in bone remodeling. Smoke induced expression of S100A8 and S100A9 in a time-dependent fashion, which was opposite of the changes in the ratio of OPG/RANKL in bone marrow cells, suggesting that the high levels of S100A8 and S100A9 may be associated with smoke-induced bone loss by increasing bone resorption.

Profiling Pancreatic Cancer–Secreted Proteome Using 15N Amino Acids and Serum-Free Media

Objectives: A new method of determining protein turnover by labeling protein with 15N amino acids was used in conjunction with serum-free cell culture to profile secreted proteins that are released by MIA PaCa-2 pancreatic cancer cells in culture. Methods: MIA PaCa-2 cells were first cultured in Dulbecco modified Eagle medium (Gibco by Invitrogen, Carlsbad, Calif) with 10% fetal bovine serum, then in serum-free modified Eagle medium with or without 50% 15N algal amino acid mixture. The effect of oxythiamine chloride on secreteome was studied. Secreteome from cell culture media was analyzed by 2-dimensional (2D) gel electrophoresis. Differentially expressed proteins were detected and identified. Protein turnover rates were calculated according to the newly established method. Western blot and enzyme-linked immunosorbent assay were used to validate identified proteins. Results: Among the 14 differentially expressed proteins after oxythiamine treatment, tissue inhibitor of metalloproteases-1 and cytokeratin-10 were identified as 2 newly synthesized secreted proteins caused by substantial 15N incorporation. The inhibition of tissue inhibitor of metalloproteases-1 expression in MIA PaCa-2 cells by oxythiamine treatment was first demonstrated by 2D gel electrophoresis and further validated by Western blotting and enzyme-linked immunosorbent assay analyses. Conclusions: Our method of labeling protein with 15N amino acids in conjunction with serum-free cell culture allows the identification of actively secreted proteins from pancreatic cancer cells and is a useful method for serum biomarker discovery.

Abstract 2025: Let-7 family miRNAs regulate estrogen signaling pathways mediated by ER alpha-66 and ER alpha-36 in breast cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC To understand how microRNAs (miRNAs) regulate breast cancer tumorigenesis, a miRNA expression microarray screening was performed using RNAs from formalin-fixed paraffin-embedded (FFPE) breast tissues, which included benign (n=13), ductal carcinoma in situ (DCIS) (n=16), and invasive ductal carcinoma (IDC) (n=15). Twenty miRNAs that were differentially expressed (p<0.01) were identified, of which let-7 family miRNAs were down-regulated in human breast cancer tissues at stages of DCIS and IDC compared to benign stage. To understand the role of let-7 miRNAs further, we performed bioinformatics analysis and found that let-7 miRNA sequences match sequences in the 3’-UTRs of estrogen receptor alpha 66 (ER-α66) and another novel receptor ER-α36. The targeting of let-7 miRNAs on ER-α66 and ER-α36 was further confirmed by a number of experimental assays, including luciferase assay, protein expression, and mRNA expression. Overexpression of let-7 miRNAs in ER-α66-positive breast cancer MCF7 cell line negatively affected ER-α66 mediated genomic estrogen pathway. As expected, down-regulation of the ER-α66 signaling by let-7 miRNAs inhibited cell proliferation, and subsequently triggered the cell apoptotic process in MCF7 cells. On the other hand, overexpression of let-7 miRNAs in ER-α66-negative cell line MB-MDA-231 negatively affected ER-α36 mediated non-genomic estrogen pathway. Besides, down-regulation of let-7 is correlated with up-regulation of ER-α36 in tamoxifen (Tam) and ICI-182780 (ICI) resistance in MCF7 cell line. Forced overexpression in Tam-and ICI-resistant MCF7 cells dampened ER-α36 expression. In conclusion, our findings not only indicate a new regulatory mechanism of let-7 miRNAs in ER mediated cellular malignant growth of breast cancer, and but also provide potential biomarkers and/or surrogate therapeutic targets useful for early diagnosis and/or therapeutic options for breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2025.

Abstract P3-09-05: Let-7 microRNAs Regulate a Novel Variant of Estrogen Receptor Alpha, ER-≥36, Nongenomic Estrogen Signal Pathway, and Tamoxifen Resistance in Breast Cancer

Background: MicroRNAs (miRNAs) have important regulatory functions in breast cancer tumorigenesis. We previously found that let-7 miRNAs were significantly downregulated in formalin-fixed paraffin-embedded (FFPE) breast cancer tissues. Material and Methods: QRT-PCR was used to check let-7 family miRNAs in FFPE tissues and breast cancer cell lines as well as expression of estrogen receptor (ER)-≥36, a variant of ER-≥66, after let-7 miRNA transfecton. Immunoblot analysis was employed to check protein expression in FFPE tissue and breast cancer cell lines. Luciferase reporter assay was used to detect direct regulation of let-7 miRNA on ER-α expression. MTT assay was applied for cell proliferation after transfection of let-7 miRNAs. Results: We found that there was an inverse correlation between the expression of ER-≥36 and several members of let-7 family miRNAs in the FFPE tissue set. Let-7 miRNA sequences match sequence in the 3’ untranslated region (3’ UTR) of ER-≥36, indicating ER-≥36 may be a target of let-7. Cotransfection of let-7 mimics with ER-≥36 3’ UTR luciferase construct decreased the activity of reporter gene. Conversely, let-7 inhibitors enhanced the reporter gene activity. Transfection of let-7 mimics inhibited both the mRNA and protein levels of ER-≥36 and further inhibited the non-genomic estrogen pathway mediated by ER-≥36 in MDA-MB-231 cells. On the contrary, transfection of let-7 inhibitors enhanced the ER-≥36 expression at both mRNA and protein levels in 184A1 cells. The high expression of ER-≥36 in tamoxifen resistant MCF7 cells can be inhibited by transfection of let-7 mimics and sensitivity toward tamoxifen is enhanced. Conclusion: Let-7 miRNAs regulate the expression of ER-≥36, which is involved in non-genomic estrogen pathway and tamoxifen resistance. Let-7 could be therapeutic target for breast cancer treatment. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-09-05.