The unique transcriptional response produced by concurrent estrogen and progesterone treatment in breast cancer cells results in upregulation of growth factor pathways and switching from a Luminal A to a Basal-like subtype.

BACKGROUND: In breast cancer, progesterone receptor (PR) positivity or abundance is positively associated with survival and treatment response. It was initially believed that PR was a useful diagnostic marker of estrogen receptor activity, but increasingly PR has been recognised to play an important biological role in breast homeostasis, carcinogenesis and metastasis. Although PR expression is almost exclusively observed in estrogen receptor positive tumors, few studies have investigated the cellular mechanisms of PR action in the context of ongoing estrogen signalling. METHODS: In this study, we contrast PR function in estrogen pretreated ZR-75-1 breast cancer cells with vehicle treated ZR-75-1 and T-47D breast cancer cells using expression microarrays and chromatin immunoprecipitation-sequencing. RESULTS: Estrogen cotreatment caused a dramatic increase in the number of genes regulated by progesterone in ZR-75-1 cells. In T-47D cells that have naturally high levels of PR, estrogen and progesterone cotreatment resulted in a reduction in the number of regulated genes in comparison to treatment with either hormone alone. At a genome level, estrogen pretreatment of ZR-75-1 cells led to a 10-fold increase in the number of PR DNA binding sites detected using ChIP-sequencing. Time course assessment of progesterone regulated genes in the context of estrogen pretreatment highlighted a series of important regulatory pathways, including those driven by epithelial growth factor receptor (EGFR). Importantly, progesterone applied to cells pretreated with estradiol resulted in switching of the PAM50-determined intrinsic breast cancer subtype from Luminal A to Basal-like, and increased the Oncotype DX® Unscaled Recurrence Score. CONCLUSION: Estrogen pretreatment of breast cancer cells increases PR steady state levels, resulting in an unequivocal progesterone response that upregulates key members of growth factor pathways. The transformative changes progesterone exerts on the breast cancer subtype suggest that these subtyping tools should be used with caution in premenopausal women.

Molecular and structural basis of androgen receptor responses to dihydrotestosterone, medroxyprogesterone acetate and Δ(4)-tibolone.

Medroxyprogesterone acetate (MPA) has widely been used in hormone replacement therapy (HRT), and is associated with an increased risk of breast cancer, possibly due to disruption of androgen receptor (AR) signaling. In contrast, the synthetic HRT Tibolone does not increase breast density, and is rapidly metabolized to estrogenic 3α-OH-tibolone and 3ß-OH-tibolone, and a delta-4 isomer (Δ(4)-TIB) that has both androgenic and progestagenic properties. Here, we show that 5α-dihydrotestosterone (DHT) and Δ(4)-TIB, but not MPA, stabilize AR protein levels, initiate specific AR intramolecular interactions critical for AR transcriptional regulation, and increase proliferation of AR positive MDA-MB-453 breast cancer cells. Structural modeling and molecular dynamic simulation indicate that Δ(4)-TIB induces a more stable AR structure than does DHT, and MPA a less stable one. Microarray expression analyses confirms that the molecular actions of Δ(4)-TIB more closely resembles DHT in breast cancer cells than either ligand does to MPA.

Diamide linked γ-cyclodextrin dimers as molecular-scale delivery systems for the medicinal pigment curcumin to prostate cancer cells.

Diamide linked γ-cyclodextrin (γ-CD) dimers are proposed as molecular-scale delivery agents for the anticancer agent curcumin. N,N'-Bis(6(A)-deoxy-γ-cyclodextrin-6(A)-yl)succinamide (66γCD2su) and N,N'-bis(6(A)-deoxy-γ-cyclodextrin-6(A)-yl)urea (66γCD2ur) markedly suppress the degradation of curcumin by forming a strong 1:1 cooperative binding complexes. The results presented in this study describe the potential efficacy of 66γCD2su and 66γCD2ur for intracellular curcumin delivery to cancer cells. Cellular viability assays demonstrated a dose-dependent antiproliferative effect of curcumin in human prostate cancer (PC-3) cells that was preserved by the curcumin-66γCD2su complex. In contrast, delivery of curcumin by 66γCD2ur significantly delayed the antiproliferative effect. We observed similar patterns of gene regulation in PC-3 cells for curcumin complexed with either 66γCD2su or 66γCD2ur in comparison to curcumin alone, although curcumin delivered by either 66γCD2su or 66γCD2ur induces a slightly higher up-regulation of heme oxygenase-1. Highlighting their nontoxic nature, neither 66γCD2su nor 66γCD2ur carriers alone had any measurable effect on cell proliferation or candidate gene expression in PC-3 cells. Finally, confocal fluorescence imaging and uptake studies were used to demonstrate the intracellular delivery of curcumin by 66γCD2su and 66γCD2ur. Overall, these results demonstrate effective intracellular delivery and action of curcumin when complexed with 66γCD2su and 66γCD2ur, providing further evidence of their potential applications to deliver curcumin effectively in cancer and other treatment settings.

Palmitoylation-dependent plasma membrane transport but lipid raft-independent signaling by linker for activation of T cells.

Linker for activation of T cells (LAT) is a dually palmitoylated transmembrane adaptor protein essential for T cell development and activation. However, whether LAT palmitoylation and/or lipid raft localization are required for its function is controversial. To address this question, we used a combination of biochemical, imaging, and genetic approaches, including LAT retrovirus-transduced mouse T cells and bone marrow chimeric mice. A nonpalmitoylated, non-lipid raft-residing mutant of transmembrane LAT could not reconstitute T cell development in bone marrow chimeric mice. This mutant was absent from the plasma membrane (PM) and was restricted mainly to the Golgi apparatus. A chimeric, nonpalmitoylated LAT protein consisting of the PM-targeting N-terminal sequence of Src kinase and the LAT cytoplasmic domain (Src-LAT) localized as a peripheral membrane protein in the PM, but outside lipid rafts. Nevertheless, Src-LAT restored T cell development and activation. Lastly, monopalmitoylation of LAT on Cys(26) (but not Cys(29)) was required and sufficient for its PM transport and function. Thus, the function of LAT in T cells requires its PM, but not raft, localization, even when expressed as a peripheral membrane protein. Furthermore, LAT palmitoylation functions primarily as a sorting signal required for its PM transport.

Impaired activation and localization of LAT in anergic T cells as a consequence of a selective palmitoylation defect.

The molecular basis of T cell anergy is not completely understood. We show that in antigen-primed anergic murine CD4(+) T cells the linker for activation of T cells (LAT) is hypophosphorylated upon CD3/CD28 restimulation. Signaling events downstream of LAT (PLCgamma1 phosphorylation and p85 [PI3-K] association) were impaired, whereas upstream events (CD3zeta and ZAP-70 phosphorylation) remained intact. LAT recruitment to the immunological synapse and its localization in detergent-resistant membrane (DRM) fractions were defective in anergic T cells. These defects resulted from impaired palmitoylation of LAT and were selective since the DRM localization and palmitoylation of Fyn were intact. This LAT defect was independent of Cbl-b and did not reflect enhanced LAT degradation. These results identify LAT as the most upstream target of anergy induction; moreover, they suggest that regulation of the amount of LAT in the immunological synapse and DRM by posttranslational palmitoylation contributes to the induction of T cell anergy.