Min Ni

ER chaperones in mammalian development and human diseases.

The field of endoplasmic reticulum (ER) stress in mammalian cells has expanded rapidly during the past decade, contributing to understanding of the molecular pathways that allow cells to adapt to perturbations in ER homeostasis. One major mechanism is mediated by molecular ER chaperones which are critical not only for quality control of proteins processed in the ER, but also for regulation of ER signaling in response to ER stress. Here, we summarized the properties and functions of GRP78/BiP, GRP94/gp96, GRP170/ORP150, GRP58/ERp57, PDI, ERp72, calnexin, calreticulin, EDEM, Herp and co‐chaperones SIL1 and P58IPK and their role in development and diseases. Many of the new insights are derived from recently constructed mouse models where the genes encoding the chaperones are genetically altered, providing invaluable tools for examining the physiological involvement of the ER chaperones in vivo.

PIF3, a Phytochrome-Interacting Factor Necessary for Normal Photoinduced Signal Transduction, Is a Novel Basic Helix-Loop-Helix Protein

The mechanism by which the phytochrome (phy) photoreceptor family transduces informational light signals to photoresponsive genes is unknown. Using a yeast two-hybrid screen, we have identified a phytochrome-interacting factor, PIF3, a basic helix-loop-helix protein containing a PAS domain. PIF3 binds to wild-type C-terminal domains of both phyA and phyB, but less strongly to signaling-defective, missense mutant-containing domains. Expression of sense or antisense PIF3 sequences in transgenic Arabidopsis perturbs photoresponsiveness in a manner indicating that PIF3 functions in both phyA and phyB signaling pathways in vivo. PIF3 localized to the nucleus in transient transfection experiments, indicating a potential role in controlling gene expression. Together, the data suggest that phytochrome signaling to photoregulated genes includes a direct pathway involving physical interaction between the photoreceptor and a transcriptional regulator.

Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light

The phytochrome photoreceptor family directs plant gene expression by switching between biologically inactive and active conformers in response to the sequential absorption of red and far-red photons,. Several intermediates that act late in the phytochrome signalling pathway have been identified, but fewer have been identified that act early in the pathway,. We have cloned a nuclear basic helix–loop–helix protein, PIF3, which can bind to non-photoactive carboxy-terminal fragments of phytochromes A and B and functions in phytochrome signalling in vivo. Here we show that full-length photoactive phytochrome B binds PIF3 in vitro only upon light-induced conversion to its active form, and that photoconversion back to its inactive form causes dissociation from PIF3. We conclude that photosensory signalling by phytochrome B involves light-induced, conformer-specific recognition of the putative transcriptional regulator PIF3, providing a potential mechanism for direct photoregulation of gene expression.

Nucleosome Dynamics Define Transcriptional Enhancers

Chromatin plays a central role in eukaryotic gene regulation. We performed genome-wide mapping of epigenetically marked nucleosomes to determine their position both near transcription start sites and at distal regulatory elements, including enhancers. In prostate cancer cells, where androgen receptor binds primarily to enhancers, we found that androgen treatment dismisses a central nucleosome present at androgen receptor binding sites that is flanked by a pair of marked nucleosomes. A new quantitative model built on the behavior of such nucleosome pairs correctly identified regions bound by the regulators of the immediate androgen response, including androgen receptor and FOXA1. More importantly, this model also correctly predicted previously unidentified binding sites for other transcription factors present after prolonged androgen stimulation, including OCT1 and NKX3-1. Therefore, quantitative modeling of enhancer structure provides a powerful predictive method to infer the identity of transcription factors involved in cellular responses to specific stimuli.

Beyond the endoplasmic reticulum: Atypical GRP78 in cell viability, signalling and therapeutic targeting

GRP78 (glucose-regulated protein of 78 kDa) is traditionally regarded as a major ER (endoplasmic reticulum) chaperone facilitating protein folding and assembly, protein quality control, Ca(2+) binding and regulating ER stress signalling. It is a potent anti-apoptotic protein and plays a critical role in tumour cell survival, tumour progression and angiogenesis, metastasis and resistance to therapy. Recent evidence shows that GRP78 can also exist outside the ER. The finding that GRP78 is present on the surface of cancer but not normal cells in vivo represents a paradigm shift on how GRP78 controls cell homoeostasis and provides an opportunity for cancer-specific targeting. Cell-surface GRP78 has emerged as an important regulator of tumour cell signalling and viability as it forms complexes with a rapidly expanding repertoire of cell-surface protein partners, regulating proliferation, PI3K (phosphoinositide 3-kinase)/Akt signalling and cell viability. Evidence is also emerging that GRP78 serves as a receptor for viral entry into host cells. Additionally, a novel cytosolic form of GRP78 has been discovered prominently in leukaemia cells. These, coupled with reports of nucleus- and mitochondria-localized forms of GRP78, point to the previously unanticipated role of GRP78 beyond the ER that may be critical for cell viability and therapeutic targeting.

Critical Role of the Stress Chaperone GRP78/BiP in Tumor Proliferation, Survival, and Tumor Angiogenesis in Transgene-Induced Mammary Tumor Development

The unfolded protein response (UPR) is an evolutionarily conserved mechanism that activates both proapoptotic and survival pathways to allow eukaryotic cells to adapt to endoplasmic reticulum (ER) stress. Although the UPR has been implicated in tumorigenesis, its precise role in endogenous cancer remains unclear. A major UPR protective response is the induction of the ER chaperone GRP78/BiP, which is expressed at high levels in a variety of tumors and confers drug resistance in both proliferating and dormant cancer cells. To determine the physiologic role of GRP78 in in situ-generated tumor and the consequence of its suppression on normal organs, we used a genetic model of breast cancer in the Grp78 heterozygous mice where GRP78 expression level was reduced by about half, mimicking anti-GRP78 agents that achieve partial suppression of GRP78 expression. Here, we report that Grp78 heterozygosity has no effect on organ development or antibody production but prolongs the latency period and significantly impedes tumor growth. Our results reveal three major mechanisms mediated by GRP78 for cancer progression: enhancement of tumor cell proliferation, protection against apoptosis, and promotion of tumor angiogenesis. Importantly, although partial reduction of GRP78 in the Grp78 heterozygous mice substantially reduces the tumor microvessel density, it has no effect on vasculature of normal organs. Our findings establish that a key UPR target GRP78 is preferably required for pathophysiologic conditions, such as tumor proliferation, survival, and angiogenesis, underscoring its potential value as a novel therapeutic target for dual antitumor and antiangiogenesis activity.

The unfolded protein response regulator GRP78/BiP is required for endoplasmic reticulum integrity and stress-induced autophagy in mammalian cells

In mammalian cells, endoplasmic reticulum (ER) stress has recently been shown to induce autophagy and the induction requires the unfolded protein response (UPR) signaling pathways. However, little is known whether autophagy regulates UPR pathways and how specific UPR targets might control autophagy. Here, we demonstrated that although ER stress-induced autophagy was suppressed by class III phosphatidylinositol-3′-kinase (PI3KC3) inhibitor 3-methyladenine (3-MA), wortmannin and knockdown of Beclin1 using small interfering RNA (siRNA), only 3-MA suppressed UPR activation. We discovered that the UPR regulator and ER chaperone GRP78/BiP is required for stress-induced autophagy. In cells in which GRP78 expression was knocked down by siRNA, despite spontaneous activation of UPR pathways and LC3 conversion, autophagosome formation induced by ER stress as well as by nutrition starvation was inhibited. GRP78 knockdown did not disrupt PI3KC3–Beclin1 association. However, electron microscopic analysis of the intracellular organelle structure reveals that the ER, a putative membrane source for generating autophagosomal double membrane, was massively expanded and disorganized in cells in which GRP78 was knocked down. ER expansion is known to be dependent on the UPR transcription factor XBP-1. Simultaneous knockdown of GRP78 and XBP-1 recovered normal levels of stress-induced autophagosome formation. Thus, these studies uncover 3-MA as an inhibitor of UPR activation and establish GRP78 as a novel obligatory component of autophagy in mammalian cells.

XBP1 promotes triple-negative breast cancer by controlling the HIF1α pathway

Cancer cells induce a set of adaptive response pathways to survive in the face of stressors due to inadequate vascularization. One such adaptive pathway is the unfolded protein (UPR) or endoplasmic reticulum (ER) stress response mediated in part by the ER-localized transmembrane sensor IRE1 (ref. 2) and its substrate XBP1 (ref. 3). Previous studies report UPR activation in various human tumours, but the role of XBP1 in cancer progression in mammary epithelial cells is largely unknown. Triple-negative breast cancer (TNBC)—a form of breast cancer in which tumour cells do not express the genes for oestrogen receptor, progesterone receptor and HER2 (also called ERBB2 or NEU)—is a highly aggressive malignancy with limited treatment options. Here we report that XBP1 is activated in TNBC and has a pivotal role in the tumorigenicity and progression of this human breast cancer subtype. In breast cancer cell line models, depletion of XBP1 inhibited tumour growth and tumour relapse and reduced the CD44highCD24low population. Hypoxia-inducing factor 1α (HIF1α) is known to be hyperactivated in TNBCs. Genome-wide mapping of the XBP1 transcriptional regulatory network revealed that XBP1 drives TNBC tumorigenicity by assembling a transcriptional complex with HIF1α that regulates the expression of HIF1α targets via the recruitment of RNA polymerase II. Analysis of independent cohorts of patients with TNBC revealed a specific XBP1 gene expression signature that was highly correlated with HIF1α and hypoxia-driven signatures and that strongly associated with poor prognosis. Our findings reveal a key function for the XBP1 branch of the UPR in TNBC and indicate that targeting this pathway may offer alternative treatment strategies for this aggressive subtype of breast cancer.

Transcriptional silencing of γ-globin by BCL11A involves long-range interactions and cooperation with SOX6

The developmental switch from human fetal (gamma) to adult (beta) hemoglobin represents a clinically important example of developmental gene regulation. The transcription factor BCL11A is a central mediator of gamma-globin silencing and hemoglobin switching. Here we determine chromatin occupancy of BCL11A at the human beta-globin locus and other genomic regions in vivo by high-resolution chromatin immunoprecipitation (ChIP)-chip analysis. BCL11A binds the upstream locus control region (LCR), epsilon-globin, and the intergenic regions between gamma-globin and delta-globin genes. A chromosome conformation capture (3C) assay shows that BCL11A reconfigures the beta-globin cluster by modulating chromosomal loop formation. We also show that BCL11A and the HMG-box-containing transcription factor SOX6 interact physically and functionally during erythroid maturation. BCL11A and SOX6 co-occupy the human beta-globin cluster along with GATA1, and cooperate in silencing gamma-globin transcription in adult human erythroid progenitors. These findings collectively demonstrate that transcriptional silencing of gamma-globin genes by BCL11A involves long-range interactions and cooperation with SOX6. Our findings provide insight into the mechanism of BCL11A action and new clues for the developmental gene regulatory programs that function at the beta-globin locus.

Targeting androgen receptor in estrogen receptor-negative breast cancer.

Endocrine therapies for breast cancer that target the estrogen receptor (ER) are ineffective in the 25%-30% of cases that are ER negative (ER-). Androgen receptor (AR) is expressed in 60%-70% of breast tumors, independent of ER status. How androgens and AR regulate breast cancer growth remains largely unknown. We find that AR is enriched in ER- breast tumors that overexpress HER2. Through analysis of the AR cistrome and androgen-regulated gene expression in ER-/HER2+ breast cancers we find that AR mediates ligand-dependent activation of Wnt and HER2 signaling pathways through direct transcriptional induction of WNT7B and HER3. Specific targeting of AR, Wnt or HER2 signaling impairs androgen-stimulated tumor cell growth suggesting potential therapeutic approaches for ER-/HER2+ breast cancers.