Elaine T. Alarid

Biological implications of polydimethylsiloxane-based microfluidic cell culture

Polydimethylsiloxane (PDMS) has become a staple of the microfluidics community by virtue of its simple fabrication process and material attributes, such as gas permeability, optical transparency, and flexibility. As microfluidic systems are put toward biological problems and increasingly utilized as cell culture platforms, the material properties of PDMS must be considered in a biological context. Two properties of PDMS were addressed in this study: the leaching of uncured oligomers from the polymer network into microchannel media, and the absorption of small, hydrophobic molecules (i.e. estrogen) from serum-containing media into the polymer bulk. Uncured PDMS oligomers were detectable via MALDI-MS in microchannel media both before and after Soxhlet extraction of PDMS devices in ethanol. Additionally, PDMS oligomers were identified in the plasma membranes of NMuMG cells cultured in PDMS microchannels for 24 hours. Cells cultured in extracted microchannels also contained a detectable amount of uncured PDMS. It was shown that MCF-7 cells seeded directly on PDMS inserts were responsive to hydrophilic prolactin but not hydrophobic estrogen, reflecting its specificity for absorbing small, hydrophobic molecules; and the presence of PDMS floating in wells significantly reduced cellular response to estrogen in a serum-dependent manner. Quantification of estrogen via ELISA revealed that microchannel estrogen partitioned rapidly into the surrounding PDMS to a ratio of approximately 9:1. Pretreatments such as blocking with serum or pre-absorbing estrogen for 24 hours did not affect estrogen loss from PDMS-based microchannels. These findings highlight the importance of careful consideration of culture system properties when determining an appropriate environment for biological experiments.

Differential Regulation of Estrogen-Inducible Proteolysis and Transcription by the Estrogen Receptor α N Terminus

ABSTRACT The ubiquitin-proteasome pathway has emerged as an important regulatory mechanism governing the activity of several transcription factors. While estrogen receptor α (ERα) is also subjected to rapid ubiquitin-proteasome degradation, the relationship between proteolysis and transcriptional regulation is incompletely understood. Based on studies primarily focusing on the C-terminal ligand-binding and AF-2 transactivation domains, an assembly of an active transcriptional complex has been proposed to signal ERα proteolysis that is in turn necessary for its transcriptional activity. Here, we investigated the role of other regions of ERα and identified S118 within the N-terminal AF-1 transactivation domain as an additional element for regulating estrogen-induced ubiquitination and degradation of ERα. Significantly, different S118 mutants revealed that degradation and transcriptional activity of ERα are mechanistically separable functions of ERα. We find that proteolysis of ERα correlates with the ability of ERα mutants to recruit specific ubiquitin ligases regardless of the recruitment of other transcription-related factors to endogenous model target genes. Thus, our findings indicate that the AF-1 domain performs a previously unrecognized and important role in controlling ligand-induced receptor degradation which permits the uncoupling of estrogen-regulated ERα proteolysis and transcription.

Increases in estrogen receptor-α concentration in breast cancer cells promote serine 118/104/106-independent AF-1 transactivation and growth in the absence of estrogen

A common phenotype in breast cancer is the expansion of the estrogen receptor‐α (ER+) cell population and an inappropriate elevation of ERα protein, the latter predisposing patients for a poorer prognosis than those with lower levels of the receptor. A tetracycline‐inducible ERα overexpression model was developed in the MCF‐7 cell line to assess induction of endogenous gene activation and growth in response to elevations in ERα protein. Heightened levels of ERα resulted in aberrant promoter occupancy and gene activation in the absence of hormone, which was independent of ligand and AF‐2 function. This increased receptor activity required the amino‐terminal A/B domain and was not inhibited by tamoxifen, which supports an enhancement of AF‐1 function, yet was independent of serine‐104, 106, and 118 phosphorylation. Ligand‐independent transcription was accompanied by an increase in growth in the absence of hormonal stimulation. The results suggest that elevated levels of ERα in breast cancer cells can result in activation of receptor transcriptional function in a manner distinct from classical mechanisms that involve ligand binding or growth factor‐induced phosphorylation. Further, they describe a potential mechanism whereby increases in ERα concentration may provide a proliferative advantage by augmenting ERα function regardless of ligand status.—Fowler, A. M., Solodin, N., Preisler‐Mashek, M. T., Zhang, P., Lee, A. V., Alarid, E. T. Increases in estrogen receptor‐α concentration in breast cancer cells promote serine 118/104/106‐independent AF‐1 transactivation and growth in the absence of estrogen. FASEB J. 18, 81–93 (2004)

Repression of ESR1 through actions of estrogen receptor alpha and Sin3A at the proximal promoter.

ABSTRACT Gene expression results from the coordinated actions of transcription factor proteins and coregulators. Estrogen receptor alpha (ERα) is a ligand-activated transcription factor that can both activate and repress the expression of genes. Activation of transcription by estrogen-bound ERα has been studied in detail, as has antagonist-induced repression, such as that which occurs by tamoxifen. How estrogen-bound ERα represses gene transcription remains unclear. In this report, we identify a new mechanism of estrogen-induced transcriptional repression by using the ERα gene, ESR1. Upon estrogen treatment, ERα is recruited to two sites on ESR1, one distal (ENH1) and the other at the proximal (A) promoter. Coactivator proteins, namely, p300 and AIB1, are found at both ERα-binding sites. However, recruitment of the Sin3A repressor, loss of RNA polymerase II, and changes in histone modifications occur only at the A promoter. Reduction of Sin3A expression by RNA interference specifically inhibits estrogen-induced repression of ESR1. Furthermore, an estrogen-responsive interaction between Sin3A and ERα is identified. These data support a model of repression wherein actions of ERα and Sin3A at the proximal promoter can overcome activating signals at distal or proximal sites and ultimately decrease gene expression.

Interplay between the levels of estrogen and estrogen receptor controls the level of the granzyme inhibitor, proteinase inhibitor 9 and susceptibility to immune surveillance by natural killer cells

Estrogens promote cell proliferation and metastases in several human cancers. Here, we describe a different action of estrogens likely to contribute to tumor development-blocking immunosurveillance. In breast cancer cells, increasing concentrations of estrogen induce increasing levels of the granzyme B inhibitor, SerpinB9/proteinase inhibitor 9 (PI-9) and progressively block cell death induced by NK92 natural killer (NK) cells, but do not block killing by a second NK cell line, NKL cells. RNA interference knockdown of PI-9 abolishes estrogens ability to block NK92 cell-induced cytotoxicity. Expressing elevated levels of estrogen receptor α (ERα) increases the induced level of PI-9, and makes tamoxifen (TAM), but not raloxifene or ICI 182,780, a potent inducer of PI-9. At elevated levels of ERα, induction of PI-9 by estradiol or TAM blocks killing by both NK92 and NKL cells. When the Erk pathway is activated with epidermal growth factor, the concentration of estrogen required to induce a protective level of PI-9 is reduced to 10 pM. Elevated concentrations of estrogen and ER may provide a dual selective advantage to breast cancer cells by controlling PI-9 levels and thereby blocking immunosurveillance. Expressing elevated levels of ERα reveals a potentially important difference in the effects of TAM, raloxifene and ICI 182,780 on immunosurveillance in breast cancer.

Temporal variation in estrogen receptor-α protein turnover in the presence of estrogen

Estrogen receptor-a (ERa) is essential in the maintenance of cellular responsiveness to estrogen in the reproductive system. It is established that ligand binding induces downregulation of ERa protein by targeting receptor for destruction by the 26S proteasome. However, ERa is preserved in cells chronically exposed to estrogen and it is unknown how receptor levels are maintained in the continued presence of the signal that induces degradation. A modified pulsechase analysis was developed using a tet-inducible ERa expression system to determine the rate of ERa protein decay following both acute and chronic estrogen treatments. Upon initial hormone treatment, ERa half-life is shortened from 3 to 1 h. However, ERa half-life increases over time, achieving a half-life of w6 h in 72 h of estrogen treatment. Analysis of ERa half-life in the presence and absence of proteasome inhibitor, MG132, revealed that the increased stability is due in part to a decreased rate of proteolysis. In addition, we observed a time-dependent increase in phospho-S118 ERa and showed that the half-life of the phosphomimetic ERa mutant, S118E-ER, is identical to that of wild-type receptor under conditions of chronic estrogen treatment. These data provide evidence that as cells adapt to chronic stimulation, ERa protein is stabilized due first to a decreased rate of proteolysis, and secondarily, to the accumulation of proteasome-resistant, phosphorylated form of receptor. This temporal control of proteolysis allows for the establishment of steady-state levels of receptor and provides a protective mechanism against loss of hormone responsiveness.

Regulation of Estrogen Receptor α N-Terminus Conformation and Function by Peptidyl Prolyl Isomerase Pin1

ABSTRACT Estrogen receptor alpha (ERα), a key driver of growth in the majority of breast cancers, contains an unstructured transactivation domain (AF1) in its N terminus that is a convergence point for growth factor and hormonal activation. This domain is controlled by phosphorylation, but how phosphorylation impacts AF1 structure and function is unclear. We found that serine 118 (S118) phosphorylation of the ERα AF1 region in response to estrogen (agonist), tamoxifen (antagonist), and growth factors results in recruitment of the peptidyl prolyl cis/trans isomerase Pin1. Phosphorylation of S118 is critical for Pin1 binding, and mutation of S118 to alanine prevents this association. Importantly, Pin1 isomerizes the serine118-proline119 bond from a cis to trans isomer, with a concomitant increase in AF1 transcriptional activity. Pin1 overexpression promotes ligand-independent and tamoxifen-inducible activity of ERα and growth of tamoxifen-resistant breast cancer cells. Pin1 expression correlates with proliferation in ERα-positive rat mammary tumors. These results establish phosphorylation-coupled proline isomerization as a mechanism modulating AF1 functional activity and provide insight into the role of a conformational switch in the functional regulation of the intrinsically disordered transactivation domain of ERα.

Purification of cell subpopulations via immiscible filtration assisted by surface tension (IFAST)

The selective isolation of a sub-population of cells from a larger, mixed population is a critical preparatory process to many biomedical assays. Here, we present a new cell isolation platform with a unique set of advantages over existing devices. Our technology, termed Immiscible Filtration Assisted by Surface Tension, exploits physical phenomena associated with the microscale to establish fluidic barriers composed of immiscible liquids. By attaching magnetically-responsive particles to a target cell population via immunocapture, we can selectively transport this population across the immiscible barrier and into a separate aqueous solution. The high interfacial energy associated with the immiscible phase / aqueous phase boundaries prevents unwanted cells or other contaminants from inadvertently crossing the immiscible phase. We have demonstrated, using fluorescent particles, stromal cells, and whole blood as “background”, that we can successfully isolate ~70% of a target breast cancer cell population with an average purity of >80%. Increased purity was obtained by coupling two immiscible barriers in series, a modification that only slightly increases operational complexity. Furthermore, several samples can be processed in parallel batches in a near-instantaneous manner without the requirement of any washing, which can cause dilution (negative selection) or significant uncontrolled loss (positive selection) of target cells. Finally, cells were observed to remain viable and proliferative following traverse through the immiscible phase, indicating that this process is suitable for a variety of downstream assays, including those requiring intact living cells.

Pin1 modulates ERα levels in breast cancer through inhibition of phosphorylation-dependent ubiquitination and degradation.

Estrogen receptor-alpha (ERα) is an important biomarker used to classify and direct therapy decisions in breast cancer (BC). Both ERα protein and its transcript, ESR1, are used to predict response to tamoxifen therapy, yet certain tumors have discordant levels of ERα protein and ESR1, which is currently unexplained. Cellular ERα protein levels can be controlled post-translationally by the ubiquitin-proteasome pathway through a mechanism that depends on phosphorylation at residue S118. Phospho-S118 (pS118-ERα) is a substrate for the peptidyl prolyl isomerase, Pin1, which mediates cis-trans isomerization of the pS118-P119 bond to enhance ERα transcriptional function. Here, we demonstrate that Pin1 can increase ERα protein without affecting ESR1 transcript levels by inhibiting proteasome-dependent receptor degradation. Pin1 disrupts ERα ubiquitination by interfering with receptor interactions with the E3 ligase, E6AP, which also is shown to bind pS118-ERα. Quantitative in situ assessments of ERα protein, ESR1, and Pin1 in human tumors from a retrospective cohort show that Pin1 levels correlate with ERα protein but not to ESR1 levels. These data show that ERα protein is post-translationally regulated by Pin1 in a proportion of breast carcinomas. As Pin1 impacts both ERα protein levels and transactivation function, these data implicate Pin1 as a potential surrogate marker for predicting outcome of ERα-positive BC.

Proteasome inhibition represses ERα gene expression in ER+ cells: a new link between proteasome activity and estrogen signaling in breast cancer

Estrogen receptor-α (ERα) is a major therapeutic target of hormonal therapies in breast cancer, and its expression in tumors is predictive of clinical response. Protein levels of ERα are tightly controlled by the 26S proteasome; yet, how the clinical proteasome inhibitor, bortezomib, affects ERα regulation has not been studied. Bortezomib selectively inhibits the chymotrypsin-like activity of the proteasome. Unlike other laboratory proteasome inhibitors, bortezomib failed to stabilize ERα protein at a dose exceeding 90% inhibition of the chymotrypsin-like activity. Unexpectedly, however, chronic bortezomib exposure caused a reduction of ERα levels in multiple ER+ breast cancer cell lines. This response can be explained by the fact that bortezomib induced a dramatic decrease in ERα mRNA because of direct transcriptional inhibition and loss of RNA polymerase II recruitment on the ERα gene promoter. Bortezomib treatment resulted in promoter-specific changes in estrogen-induced gene transcription that related with occupancy of ERα and RNA polymerase II (PolII) on endogenous promoters. In addition, bortezomib inhibited estrogen-dependent growth in soft agar. These results reveal a novel link between proteasome activity and expression of ERα in breast cancer and uncover distinct roles of the chymotrypsin-like activity of the proteasome in the regulation of the ERα pathway.