Estrogen Effects Differ Between Medium Maintenance and Replacement from Transcriptional and Clinical Perspectives in T47D Breast Cancer Cells.

BACKGROUND/AIM: Our most recent study revealed that the responsiveness of hormone receptor-positive breast cancer (HR+ BC) cells to estrogen or endocrine therapy can be altered by certain cell culture or ambient environmental conditions. Nevertheless, we were unable to investigate the relevant molecular mechanism and clinical relevance. Therefore, this study was planned as a follow-up. MATERIALS AND METHODS: RNA sequencing was mainly used with T47D cells treated with or without 17ß-estradiol (E2) under medium maintenance (MTN; conventional culture method) and medium exchange (EXC; daily replacing the existing medium with fresh medium). RESULTS: The role of E2 in transcription differed between MTN and EXC, and E2 played more important roles in transcription in terms of cancer development under EXC than under MTN, consistent with the previous functional effects of EXC. The novel concept of the "estrogen-responsive and proliferation-related gene (ERPG)" was introduced. The expression of ERPGs, which were distinguished from typical estrogen-responsive genes, was correlated with that of prognostic and predictive factors for HR+ BC. The transcriptional induction of ERPGs and typical estrogen-responsive genes regardless of E2 treatment under MTN was reminiscent of constitutive estrogen receptor (ER) activation. Additionally, phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) inhibitors were more effective under EXC than under MTN. CONCLUSION: This study, demonstrating the more important roles of estrogen in terms of cancer development under EXC than under MTN, supports the use of our research model in future studies to overcome endocrine resistance in HR+ BC.

A New Culture Model for Enhancing Estrogen Responsiveness in HR+ Breast Cancer Cells through Medium Replacement: Presumed Involvement of Autocrine Factors in Estrogen Resistance.

Hormone receptor-positive breast cancer (HR+ BC) cells depend on estrogen and its receptor, ER. Due to this dependence, endocrine therapy (ET) such as aromatase inhibitor (AI) treatment is now possible. However, ET resistance (ET-R) occurs frequently and is a priority in HR+ BC research. The effects of estrogen have typically been determined under a special culture condition, i.e., phenol red-free media supplemented with dextran-coated charcoal-stripped fetal bovine serum (CS-FBS). However, CS-FBS has some limitations, such as not being fully defined or ordinary. Therefore, we attempted to find new experimental conditions and related mechanisms to improve cellular estrogen responsiveness based on the standard culture medium supplemented with normal FBS and phenol red. The hypothesis of pleiotropic estrogen effects led to the discovery that T47D cells respond well to estrogen under low cell density and medium replacement. These conditions made ET less effective there. The fact that several BC cell culture supernatants reversed these findings implies that housekeeping autocrine factors regulate estrogen and ET responsiveness. Results reproduced in T47D subclone and MCF-7 cells highlight that these phenomena are general among HR+ BC cells. Our findings offer not only new insights into ET-R but also a new experimental model for future ET-R studies.

ADRM1 gene amplification is a candidate driver for metastatic gastric cancers.

We searched for candidate target genes in metastatic gastric cancer, using comparative genomic hybridization (CGH) and mRNA expression array analysis of endoscopic biopsy samples collected from 32 patients. Recurrent amplicons included 17q21.2 (36,569,293-37,307,055), 8q24.13-q24.21 (126,357,475-130,159,285), and 20q13.33 (60,211,249-61,382,787). In this paper, we focused on the 1.1-Mb genomic region containing 24 genes in chromosome 20q13.33 (from 60,211,249 to 61,382,787), the third most frequent amplicon that was amplified in three of 32 patients (9.4 %), with log2 tumor/reference ratios ranging from 0.6 to 1.5. Of three genes in the 20q13.33 amplicon, ADRM1 was chosen for functional analyses. ADRM1 knockdown suppressed the proliferation of two human gastric cancer cells, SNU-601 and SNU-216. Overexpression of Adrm1 promoted cell proliferation of conditionally-immortalized, mouse ImSt gastric epithelial cells, with increased S1 phase fraction and decreased expression of p21(Cip1). These results collectively indicate that ADRM1 promoted gastric epithelial cell proliferation by cell cycle progression. Therefore, ADRM1 is a candidate target gene in the chromosome 20q13.33 amplicon that may possibly be linked to development of gastric cancer.

Preclinical efficacy testing for stomach and liver cancers.

PURPOSE: Hollow fiber assays offer an early in vivo method of anticancer drug screening. The assays have been optimized for human cancers originating from the lung, breast, colon, ovary, and brain, but not from the stomach and liver. The current study focused on optimization of hollow fiber assays for gastric and hepatocellular carcinoma cell lines. MATERIALS AND METHODS: Gastric (SNU-16, SNU-484, SNU-668) and hepatocellular (HepG2, SK-Hep-1, Hep3B) carcinoma cell lines in hollow fibers were transplanted subcutaneously and intraperitoneally into mice, which were subsequently treated with a standard anticancer agent, paclitaxel. The hollow fiber activity of paclitaxel in each cell line was compared with the xenograft activity. RESULTS: Using optimized inoculation densities and schedules, treatment with paclitaxel was effective in gastric carcinoma cell lines, SNU-16 and SNU-484, but not in SNU-668. In the hollow fiber assays, paclitaxel was effective in hepatocellular carcinoma cell lines, HepG2 and SK-Hep-1, but not in Hep3B. Consistent with the results of the hollow fiber assay, SNU-16 and SNU-484, but not SNU-668, showed tumor regression, and HepG2 and SK-Hep-1, but not Hep3B, showed effective tumor responses following treatment with paclitaxel in xenograft models. When EW7197, a novel compound, and flavopiridol were tested in SNU-16 cells under optimized conditions, the hollow fiber activity showed good correlation with the xenograft activity of each compound. CONCLUSION: Our protocols may be useful for screening candidate small molecules that may exhibit activity against stomach and liver cancers, both of which are common in Korea.

Cooperativity of E-cadherin and Smad4 loss to promote diffuse-type gastric adenocarcinoma and metastasis.

UNLABELLED: Loss of E-cadherin (CDH1), Smad4, and p53 has been shown to play an integral role in gastric, intestinal, and breast cancer formation. Compound conditional knockout mice for Smad4, p53, and E-cadherin were generated to define and compare the roles of these genes in gastric, intestinal, and breast cancer development by crossing with Pdx-1-Cre, Villin-Cre, and MMTV-Cre transgenic mice. Interestingly, gastric adenocarcinoma was significantly more frequent in Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(F) (/+) mice than in Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(+/+) mice, demonstrating that Cdh1 heterozygosity accelerates the development and progression of gastric adenocarcinoma, in combination with loss of Smad4 and p53. Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(F) (/+) mice developed gastric adenocarcinomas without E-cadherin expression. However, intestinal and mammary adenocarcinomas with the same genetic background retained E-cadherin expression and were phenotypically similar to mice with both wild-type Cdh1 alleles. Lung metastases were identified in Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(F) (/+) mice, but not in the other genotypes. Nuclear ß-catenin accumulation was identified at the invasive tumor front of gastric adenocarcinomas arising in Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(F) (/+) mice. This phenotype was less prominent in mice with intact E-cadherin or Smad4, indicating that the inhibition of ß-catenin signaling by E-cadherin or Smad4 downregulates signaling pathways involved in metastases in Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(F) (/+) mice. Knockdown of ß-catenin significantly inhibited the migratory activity of Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(F) (/+) cell lines. Thus, loss of E-cadherin and Smad4 cooperates with p53 loss to promote the development and metastatic progression of gastric adenocarcinomas, with similarities to human gastric adenocarcinoma. IMPLICATIONS: This study demonstrates that inhibition of ß-catenin is a converging node for the antimetastatic signaling pathways driven by E-cadherin and Smad4 in Pdx-1-Cre;Smad4(F/F);Trp53(F/F);Cdh1(F) (/+) mice, providing novel insights into mechanisms for gastric cancer metastasis.

Protein and lipid MALDI profiles classify breast cancers according to the intrinsic subtype.

BACKGROUND: Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) has been demonstrated to be useful for molecular profiling of common solid tumors. Using recently developed MALDI matrices for lipid profiling, we evaluated whether direct tissue MALDI MS analysis on proteins and lipids may classify human breast cancer samples according to the intrinsic subtype. METHODS: Thirty-four pairs of frozen, resected breast cancer and adjacent normal tissue samples were analyzed using histology-directed, MALDI MS analysis. Sinapinic acid and 2,5-dihydroxybenzoic acid/α-cyano-4-hydroxycinnamic acid were manually deposited on areas of each tissue section enriched in epithelial cells to identify lipid profiles, and mass spectra were acquired using a MALDI-time of flight instrument. RESULTS: Protein and lipid profiles distinguish cancer from adjacent normal tissue samples with the median prediction accuracy of 94.1%. Luminal, HER2+, and triple-negative tumors demonstrated different protein and lipid profiles, as evidenced by permutation P values less than 0.01 for 0.632+ bootstrap cross-validated misclassification rates with all classifiers tested. Discriminatory proteins and lipids were useful for classifying tumors according to the intrinsic subtype with median prediction accuracies of 80.0-81.3% in random test sets. CONCLUSIONS: Protein and lipid profiles accurately distinguish tumor from adjacent normal tissue and classify breast cancers according to the intrinsic subtype.

High performance organic photosensitizers for dye-sensitized solar cells.

We report highly efficient organic photosensitizers containing pi-conjugated alkoxy-substituted oligophenylenevinylene linkers with electron donor-acceptor units for dye-sensitized solar cells. TA-DM-CA showed an overall solar-to-energy conversion efficiency of 9.67% at AM 1.5 illumination (100 mW cm(-2)).