Inhibition of DNA Methylation in Acquired Tamoxifen-Resistant Breast Cancer: Cell Line Model and Clinical Implications

One out of every eight American women will develop invasive breast cancer throughout their lifetime. Approximately 70% of breast cancers are estrogen receptor alpha (ER)-positive and can therefore be treated with an anti-estrogen such as tamoxifen. Although tamoxifen treatment has been successful at reducing breast cancer death rates, nearly one-third of women treated with tamoxifen for 5 years will have disease recurrence. Therefore, it is imperative that researchers investigate the mechanisms involved in developing acquired tamoxifen resistance and identify biomarkers that are predictive of acquired resistance. DNA methylation is known to play a role in the development of breast cancer and is thought to be involved in drug resistance as well. Although genome-wide hypomethylation occurs frequently in breast cancer, gene-specific hypermethylation and a corresponding decrease in expression are known to occur in breast cancer. The primary objective of this study is to evaluate how changes in DNA methylation in response to the DNMT1 inhibitor, 5-Aza-2’-deoxycytidine, affect gene expression and cell behavior in the ER-negative cell line model of tamoxifen-resistance. In the present study, I utilize a cell line model of acquired tamoxifen resistance, TMX2-28. This cell line, along with two other tamoxifen-resistant lines, were generated by culturing MCF-7 cells in the presence of Tamoxifen (10-6 M) for 6 months. Cloning by limiting dilution lead to the discovery of three tamoxifen-resistant cell lines. Two of which retained expression of ER, TMX2-4 and TMX2-11, and one that no longer expressed ER, TMX2-28. Initial studies of genome-wide methylation with the Illumina Human Methylation 450 BeadChip found TMX2-28 is hypermethylated compared to the parental cell line, MCF-7. Specifically, promoter methylation is involved in regulating the expression of at least two genes in TMX2-28. Promoter methylation of these genes decreased in response to treatment with the DNMT-inhibitor, 5-Aza-2’-deoxycytidine, and a corresponding increase in mRNA expression was observed. Unpublished data from our lab indicate that 5-Aza-2’-deoxycytidine also decreases TMX2-28 cell proliferation. However, it is unclear how changes in methylation and gene expression induced by 5-Aza-2’-deoxycytidine affect the behavior of TMX2-28 cells. I tested the hypothesis that changes in cell behavior in response to 5-Aza-2’-deoxycytidine are caused by changes in gene expression induced by decreased promoter methylation. First I utilized the Illumina Human Methylation 450 BeadChip to study changes in DNA methylation in the parental cell line, MCF-7, as well as the three tamoxifen-resistant cell lines. I confirmed results from previous studies indicating that TMX2-28 is hypermethylated compared to the parental cell line, MCF-7. I also discovered that treatment with 5-Aza-2’-deoxycytidine results in cell line-specific changes in DNA methylation. The ER-negative, tamoxifen-resistant cell line, TMX2-28, is most sensitive to treatment with the DNMT-inhibitor. To determine the genes most likely to have altered expression after 5-Aza-2’-deoxycytidine treatment, I first identified CpG sites that are hypermethylated in TMX2-28 compared to MCF-7. Of these 37,501 CpGs, there are 707 CpGs with decreased methylation after treatment with 5-Aza-2’-deoxycytidine. This corresponds to 27 genes with changes in at least 2 CpGs located in the promoter. To determine other cell behaviors that may be affected by inhibition of methylation in TMX2-28, I conducted a 2-dimensional scratch/wound assay. I found that treatment of TMX2-28 with 5-Aza-2’-deoxycytidine inhibits 2-dimensional migration and induces detachment of cells from the monolayer. Treatment with 5-Aza-2’-deoxycytidine also decreased cell viability. One of the 27 genes I identified as differentially methylated and altered by 5-Aza-2’-deoxycytidine, TACSTD2, seemed likely to play a role in regulating these behaviors in TMX2-28. Expression of the protein encoded by this gene, tumor-associated calcium signal transducer 2, TROP2, is regulated by promoter methylation in lung cancer and TROP2 is involved in development, intracellular signaling and epithelial cancers. Expression of TROP2 in lung cancer cell lines inhibits proliferation, colony formation and blocks phosphorylation of important intracellular signaling molecules involved in growth signaling and transcription. Promoter methylation of TACSTD2 is low in MCF-7 and TROP2 expression is high while promoter methylation is high in TMX2-28 and TROP2 expression is low. This is consistent with the idea that promoter methylation is regulating expression. Therefore, I tested the hypothesis that the behavior of TMX2-28 in response to 5-Aza-2’-deoxycytidine was due to increased expression of TROP2. To test this hypothesis, I first confirmed that treatment of TMX2-28 with 5-Aza-2’-deoxycytidine decreases TACSTD2 promoter methylation and increases TROP2 expression. Next, I generated stable cell lines with increased TROP2 expression (TMX2-28-Trop2) and knocked down TROP2 expression in MCF-7 (MCF-7-Trop2-Kd). I quantified proliferation, adhesion to fibronectin and migration of these cell lines. Contrary to my predictions, expression of TROP2 in TMX2-28 did not affect proliferation and increased both adhesion to fibronectin and migration. Interestingly, knockdown of TROP2 expression in MCF-7 increased proliferation but did not alter adhesion or migration. To further evaluate the role of TROP2 in breast cancer, TACSTD2 methylation and TROP2 expression were analyzed in a total of 70 primary and recurrent clinical breast cancer samples. TACSTD2 promoter methylation was lower in the clinical samples than the cell lines. In agreement with the cell line data, methylation of the three TACSTD2 CpG sites was higher in ER-negative recurrent tumors that were initially ER-positive primary tumors. However, there was no correlation between promoter methylation and TROP2 expression in the clinical samples. In contrast to previous reports, I found no association between TROP2 expression and tumor grade or Ki67 status. Despite the small size, the clinical sample data indicate that our cell line model may be relevant for studying ER-negative, tamoxifen resistant breast cancer. Additionally, I characterized DNA methylation and protein expression patterns of several luminal and basal cytokeratins. According to the methylation status determined by the 450 BeadChip and protein expression determined by IHC for the parental cell line and the three tamoxifen-resistant cell lines, promoter methylation seems to play a role in regulating expression of p40, the basal cytokeratins CK5 and CK14, and the luminal cytokeratins CK8, CK17, CK18 and CK20. I found that MCF-7, TMX2-4 and TMX2-11 express luminal (low molecular weight) cytokeratins while TMX2-28 express a combination of luminal and basal cytokeratins. These new IHC data are consistent with previously published work from our lab and demonstrate that TMX2-28 have a mixed basal-luminal phenotype. DNA methylation of cytokeratin genes may be useful as an alternative indicator of tumor subtype and provide insight into mechanisms controlling expression of these important prognostic markers. The results presented in the present study demonstrate that DNA methylation may play an important role in ER-negative, tamoxifen resistant breast cancer. TMX2-28 cells rely on a TROP2-independent mechanism to sustain proliferative signals. TROP2 acts to promote adhesion and migration in TMX2-28. Further studies are necessary to determine the mechanism by which TROP2 increases adhesion and migration as well as the role of TROP2 in drug resistant breast cancer. Due to the high percentage of clinical samples positive for TROP2 expression, TROP2 may serve as a method for targeted drug delivery for recurrent breast cancer

Two Cryptic Species within \u3cem\u3eAstragalus cusickii\u3c/em\u3e Delimited Using Molecular Phylogenetic Techniques

Understanding the source of phenotypic variability is a challenge in the biological sciences. Variation in phenotypes is the result of variation in the genetics and environment the organism experiences, but elucidating the relative contribution of these two parameters can pose problems, especially in the field of systematics. Systematists are challenged to classify biological diversity into groups that share common ancestry. Phenotypic variation can be useful to demonstrate common ancestry, but only when the primary contributor to the variation is under strong genetic control, and thus heritable. Cusick’s milkvetch (Astragalus cusickii) is a perennial forb endemic to the intermountain west region of the United States. The species currently comprises four varieties based on subtle morphological dissimilarities, such as leaf size and density, and the size and shape of the seed pods. The taxonomic organization of the varieties of A. cusickii and related species of Astragalus were reexamined through phylogenetic analysis of nuclear, nuclear-ribosomal, and chloroplast gene regions. Maximum parsimony, maximum likelihood, Bayesian inference, the genealogical sorting index, an approximately unbiased test, and multispecies coalescent analysis were used to determine appropriate species boundaries under the phylogenetic species concept. The results support reclassification of A. cusickii var. packardiae and A. cusickii var. sterilis as separate species. Additionally, evidence suggests a chloroplast capture event may have occurred in one population of A. cusickii var. packardiae

An investigation of the influence of cutting tool surface finish upon performance parameters

The functional relationships in a qualitative sense between the parameters involved in the metal cutting operation and related costs have been understood. Alterations in the surface condition of the tool to either increase the life of the tool or reduce the cost of the tool with no sacrifice in cutting effectiveness is investigated

Local tunneling spectroscopy of the electron-doped cuprate Sm1.85Ce0.15CuO4

We present local tunneling spectroscopy in the optimally electron-doped cuprate Sm2-xCexCuO4 x=0.15. A clear signature of the superconducting gap is observed with an amplitude ranging from place to place and from sample to sample (Delta~3.5-6meV). Another spectroscopic feature is simultaneously observed at high energy above \pm 50meV. Its energy scale and temperature evolution is found to be compatible with previous photoemission and optical experiments. If interpreted as the signature of antiferromagnetic order in the samples, these results could suggest the coexistence on the local scale of antiferromagnetism and superconductivity on the electron-doped side of cuprate superconductors

Effects of electron-phonon interactions on the electron tunneling spectrum of PbS quantum dots

We present a tunnel spectroscopy study of single PbS Quantum Dots (QDs) as function of temperature and gate voltage. Three distinct signatures of strong electron-phonon coupling are observed in the Electron Tunneling Spectrum (ETS) of these QDs. In the shell-filling regime, the $8\times$ degeneracy of the electronic levels is lifted by the Coulomb interactions and allows the observation of phonon sub-bands that result from the emission of optical phonons. At low bias, a gap is observed in the ETS that cannot be closed with the gate voltage, which is a distinguishing feature of the Franck-Condon (FC) blockade. From the data, a Huang-Rhys factor in the range $S\sim 1.7 - 2.5$ is obtained. Finally, in the shell tunneling regime, the optical phonons appear in the inelastic ETS $d^2I/dV^2$.Comment: 5 pages, 5 figure

Optical Spectroscopy as a Probe of Gaps and Kinetic Electronic Energy in p- and n-type cuprates

The real part of the optical in-plane conductivity of p-- and n--type cuprates thin films at various doping levels was deduced from highly accurate reflectivity measurements. We present here a comprehensive set of optical spectral weight data as a function of the temperature $T (> T_c$), for underdoped and overdoped samples. The temperature dependence of the spectral weight is not universal. Using various cut-off frequencies for the spectral weight, we show that n--type Pr$_{2-x}$Ce$_x$CuO$_4$ and p--type Bi$_2$Sr$_2$CaCu$_2$O$_{8+\delta}$ exhibit both similarities and striking differences. The Fermi surface is closed in overdoped metallic samples. In underdoped Pr$_{2-x}$Ce$_x$CuO$_4$ samples, it clearly breaks into arcs, giving rise to a "pseudogap" signature. It is argued that such a signature is subtle in underdoped Bi$_2$Sr$_2$CaCu$_2$O$_{8+\delta}$.Comment: Proceedings M2SHTSCVIII, to appear in Physica