Browsing by Subject "ER"

In the following document, I will describe two distinct strategies that poliovirus

(PV) deploys to manage host antiviral responses. In the first section, I report on a role of

the constitutive repressor of eIF2α phosphorylation (CReP) in translation of PV and the

endoplasmic reticulum (ER)-resident chaperone binding immunoglobulin protein (BiP)

at the ER. Functional, proximity-dependent labeling and cell fractionation studies

revealed that CReP, through binding of the eukaryotic translation initiation factor eIF2α,

anchors translation initiation machinery at the ER and enables protein synthesis in this

compartment. This ER site was protected from the suppression of cytoplasmic protein

synthesis by acute stress responses. I propose that partitioning of translation initiation

machinery at the ER enables cells to maintain active translation of PV during stress.

In the second section, I report that PV 2A protease cleaves all three members of

the YTHDF protein family, cytosolic N6-methyladenosine (m6A) ‘readers’ that regulate

target mRNA fate. These cleavages occurred early during infection, and preemptive

YTHDF3 depletion enhanced viral replication. This corresponded with diminished type-

I interferon (IFN) receptor (IFNAR) expression and IFN-stimulated gene induction,

while IFN production was not significantly changed. I propose that 2A protease cleaves

YTHDF proteins, in part, to interfere with IFNAR expression and antagonize the host

antiviral response.

The estrogen receptor(ER) is a master transcriptional regulator of the breast where it plays key roles in the development and maintenance of normal breast epithelium but is also critical to the growth of luminal breast cancers. ER is also a well-defined molecular therapeutic target and anti-estrogens, such as tamoxifen, are used clinically to inhibit the mitogenic activity of ER and delay disease progression. However, despite the initial benefits to tamoxifen therapy, nearly one third of luminal breast cancer tumors eventually become resistant, limiting the therapeutic utility the drug. Mechanisms of resistance can be attributed to circumvention of ER and reliance on alternative growth pathways, or through upregulation of pathways that converge with ER to allow reactivation. Understanding the molecular determinants of resistance is a critical endeavor that demands attention in order to shape new drug developments and extend the therapeutic efficacy of anti-estrogens.

A major challenge in elucidating mechanisms of resistance is in understanding the complexities of the ER signaling program in respect to receptor occupancy and the coordinated relationship with chromatin architecture and collaborating transcription factors. This work therefore integrates the relationship between accessible chromatin, as measured by DNase-Seq, with ER occupancy and ER-mediated transcription in an in vivo derived tamoxifen resistant cell line (TamR) and a comparator group of two closely related tamoxifen sensitive cell lines. Cumulatively, these data demonstrate an enhanced role for FOXA1 in tamoxifen resistance. Specifically, FOXA1 occupancy is greatly enriched at differential DNase hypersensitive loci in TamR cells, and FOXA1 target genes are dramatically upregulated. Furthermore, expression of these target genes can be restored to MCF7 levels with siRNA directed against FOXA1. The TamR cells also have increased ER occupancy at FOXA1 overlapping sites, where ER is engaged to chromatin in a ligand-independent manner and results in enhanced activation of nearby target genes that can be repressed with the pure anti-estrogen, ICI. The increased role of FOXA1 is not due to an increase in total protein levels however and instead is manifested through increased activity.

Other clinical associations of resistance have been elucidated for which there is little to no mechanistic evidence currently available. HOXB13 has been shown to associate with tamoxifen therapy failure from differential microarray expression profiling of patients who relapsed compared to those that remained disease-free at the five year follow-up. The outcome of our studies reveals HOXB13 to downregulate GATA3 levels, which in turn leads to loss of ER function and parallel activation of inflammatory pathways.

The present study also makes use of publicly available clinical datasets to generate an integrative database of 4885 patients from 25 independent studies. Furthermore, analytical methods and functions were also developed to allow efficient use and application of the data. Access to the breast cancer meta-set and functions are made available to end users via a web interface, GeneAnalytics. Together, the breast cancer meta-set and associated access through the GeneAnalytics web sites provides novel opportunities for researchers to integrate functional studies with tumor derived expression data to further our understanding of cancer related processes.

Collectively, our findings demonstrate that the ER signaling program is modified as tumors progress to resistance by an increased role of FOXA1 to facilitate ER binding and reprogramming, and by HOXB13 to suppress the actions of ER and promote inflammatory pathways. These mechanisms highlight distinct methods of resistance and provide rational for new therapeutic approaches to extend the utility of current anti-estrogens.