<p>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.</p><p> 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. </p><p>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. </p><p>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.</p><p>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.</p> / Dissertation

Axons possess a continuous network of smooth tubular endoplasmic reticulum (ER), extending from the nuclear envelope throughout the neuron to synapses. Mutations affecting proteins with intramembrane hairpin domains that model tubular ER membrane can lead to the axon degenerative disease, hereditary spastic paraplegia (HSP). However, the extent and mechanisms by which HSP proteins contribute to axonal ER organization and dynamics are unclear. To understand these mechanisms, there is a need to visualize axonal ER in wild-type and mutant live axons. I have therefore aimed to develop these tools in Drosophila larvae and adults, and use them to visualize mutant phenotypes. Firstly, I developed a system to visualize fluorescently marked ER in individual axons in adult fly legs, and tested how this can be used to investigate the effects of loss of intramembrane hairpin HSP proteins on ER in adult legs. Secondly, known mutations affecting HSP hairpin proteins reduce the axonal ER network but not severely; I hypothesized that additional HSP ER membrane proteins might contribute to residual tubule formation; these include Arl6IP, also reported to promote ER tubule formation. I generated transgenic flies to overexpress a fluorescently tagged eGFP::Arl6IP1, and found that this fusion protein localizes within axonal ER. To study whether loss of Arl6IP1 function affects axonal ER, I tested the effects of knockdown on this compartment, but found no consistent effects. To achieve stronger loss of function, I also generated a mutant stock that lacked one of the transmembrane domains and showed a slight developmental delay in homozygous Drosophila larvae. Like mutations in a number of other HSP hairpin proteins, this lesion is homozygous viable, and further characterization of its phenotype will help elucidate how Arl6IP1 contributes to modeling the axonal ER network. In conclusion, my work shows the utility of GFP markers of axonal ER, it can facilitate faster screening for other genes that potentially regulate ER structure and for ageing phenotypes that are not apparent in larval stages, and suggests Arl6IP1 as another HSP protein with a role in axonal ER organization.

Research Doctorate - Doctor of Philosophy (PhD) / Melanoma is a skin cancer that remains a major public health problem in Australia because of its high incidence and the high morbidity and mortality associated with the disease. Melanoma has proven largely resistant to many chemotherapeutic and biological agents. Hope for a new approach in treatment of melanoma comes from the identification of the mechanisms employed in induction of apoptosis by ER stress and the possible resistance mechanisms in melanoma cells against ER stress-induced apoptosis. At the beginning of this study, little was known about the effects of ER stress on melanoma. The aim of this thesis was to elucidate the mechanisms of ER stress-induced apoptosis, the interaction between ER stress pathways and other signalling pathways in melanoma, thus to provide more information in identification of treatment approaches that will increase the sensitivity of melanoma to apoptosis induced by ER stress. Studies in Chapter 3 show that most melanoma cells are relatively resistant to ER stress-induced apoptosis except one cell line Me1007. However, inhibition of the MEK/ERK sensitizes melanoma cells to ER stress-induced apoptosis. This is mediated, at least in part, by caspase-4 activation and is associated with inhibition of the ER chaperone GRP78 expression. Moreover, inhibition of the MEK/ERK pathway reduces the level of GRP78 expression as well as its up-regulation by ER stress. Therefore, when the MEK/ERK is inhibited, caspase-4 is released from its complex with GRP78 and activated to mediated apoptosis. Chapter 4 demonstrates that up-regulation of the anti-apoptotic Bcl-2 family member Mcl-1 is one of the mechanisms critical for protection of melanoma cells against ER stress-induced apoptosis. Inhibition of Mcl-1 by siRNA renders melanoma cells sensitive to apoptosis induced by the ER stress inducers Thapsigargin (TG) or Tunicamycin (TM) mediated by PUMA and Noxa. ER stress up-regulates the BH3-only proteins PUMA and Noxa, but not Bim and BIK in melanoma cells, through transcriptional mechanisms, but the increase of Noxa but not PUMA is dependent on p53. Up-regulation of Mcl-1 is also due to increased transcription that involved the IRE1α and ATF6 signaling pathways of the unfolded protein response. In addition, activation of the MEK/ERK signaling pathway appears to be necessary for optimal up-regulation of Mcl-1. Melanoma cells are largely unresponsive to chemotherapy-induced apoptosis. Activation of the Unfolded Protein Response (UPR) by ER stress has profound effects on the sensitivity of melanoma cells to clinically relevant chemotherapeutic drugs and those in development for clinical use. In Chapter 5, the DNA-damaging drugs Cisplatin and Adriamycin, and the histone deacetylase inhibitors Suberic Bishydroxamate (SBHA) and Sodium Butyrate (NaB) further activate the UPR, indicative of induction of ER stress. The MEK inhibitors U0126 and AZD6244 reduce GRP78 expression levels; however, microtubule-targeting drugs Vincristine and Docetaxel do not change the GRP78 level. Knockdown of the IREα and ATF6 pathway of the UPR, and GRP78 by siRNA results in increased sensitivity of melanoma cells to these compounds. Studies in Chapter 6 show that treatment with either Tunicamycin (TM) or Thapsigargin (TG) selectively up-regulates TRAIL-R2 expression and enhances TRAIL-induced apoptosis in melanoma cells. This appears to be cooperatively mediated by the ATF6 and IRE1α signaling pathways and GADD153/CHOP. However, although siRNA knockdown of ATF6 or IRE1α inhibits up-regulation of TRAIL-R2, it sensitizes melanoma cells to TRAIL-induced apoptosis. Thus, it appears that regulation of TRAIL-R2 expression is not the only means by which the UPR regulates TRAIL-induced apoptosis in melanoma. The UPR may also antagonize TRAIL-induced apoptotic signaling by an intracellular mechanism(s). Study of a melanoma cell line Me1007 in Chapter 7 is the only cell line sensitive to ER stress-induced apoptosis, shows that apoptosis in this cell line is induced by ER stress via a caspase-8-mediated pathway. The high sensitivity of Me1007 to ER stress-induced apoptosis is associated with low expression levels of the apoptosis repressor with caspase recruitment domain (ARC) protein. In resistant cell lines, ARC is expressed at relatively high levels, which may effectively inhibit activation of caspase 8. Therefore, ARC appears to be critical in blocking activation of casapse-8 in melanoma cells subjected to ER stress.

Our laboratory has recently reported that lecithin:cholesterol acyltransferase (LCAT) deficient mice are hypersensitive to insulin and resistant to diet-induced obesity, particularly in the LDL receptor (LDLR) knockout background. These phenotypes are linked to hepatic endoplasmic reticulum (ER) stress, which we showed is elevated basally and highly inducible in LDLR deficient mice. While in LCAT/LDLR deficient mice, ER stress is normalized basally and its diet-induction is attenuated. Mechanistically, we show here that excess free cholesterol (FC), in part from the bile, accumulates in the ER membrane of LDLR deficient mice. In contrast, LCAT/LDLR deficient mice have reduced levels of ER membrane FC and are resistant to cholesterol diet-induced elevations, in part from increased INSIG-1 expression and cholesterol esterification by ACAT2. Our analysis has led to the first report of cholesterol-induced hepatic ER stress in vivo and the identification of ER FC levels as a critical indicator of ER stress susceptibility.

Endoplasmic reticulum (ER) stress is the condition that unfolded or misfolded proteins accumulated in the ER which leads to the solubility stress. ER can activate the unfolded protein response (UPR) to restore the ER homeostasis. JAB1 was originally identified as the coactivator of AP-1 transcription factor. JAB1 was then discovered to mediate the cyclin-dependent-kinase inhibitor p27kip1 nuclear exportation and degradation. Previous studies demonstrate that ER stress may affect the regulation of JAB1, but the mechanism is still unknown. In this study, we want to investigate how JAB1 is regulated in ER stress. We applied tunicamycin, a protein N-glycosylation inhibitor, as the ER stress inducer. Western blot and reverse transcription PCR revealed that treatment with tunicamycin for 48 hours in Hep3B induced ER stress and repressed JAB1 protein and mRNA expression. Serial deletion of the JAB1 promoter activity assay revealed that the region from -405 bp to -223 bp may be responsive in the tunicamycin-induced ER stress. Computational prediction suggested that there are several candidate factors may join the regulation of JAB1 in this region. Site-directed mutation of JAB1 promoter assay revealed that the tunicamycin-induced ER stress repressed JAB1 promoter activity through the sites at -342/-338 and -331/-327 in JAB1 promoter. Chromatin immunoprecipitation assay suggested that tunicamycin-induced ER stress repressed the JAB1 promoter activity through increasing the SP1 and DNMT3b binding to the SP1 binding sites at -342/-338 and -331/-327 in JAB1 promoter. Methylation specific PCR showed that the SP1 binding sites at -342/-338 and -331/-327 in JAB1 promoter were methylated in tunicamycin-induced ER stress. Taken together, we demonstrated that tunicamycin-induced ER stress repressed the JAB1 gene expression in Hep3B through increasing the binding of SP1 and DNMT3b to the SP1 binding sites and inducing promoter methylation to repress JAB1 expression.

Our laboratory has recently reported that lecithin:cholesterol acyltransferase (LCAT) deficient mice are hypersensitive to insulin and resistant to diet-induced obesity, particularly in the LDL receptor (LDLR) knockout background. These phenotypes are linked to hepatic endoplasmic reticulum (ER) stress, which we showed is elevated basally and highly inducible in LDLR deficient mice. While in LCAT/LDLR deficient mice, ER stress is normalized basally and its diet-induction is attenuated. Mechanistically, we show here that excess free cholesterol (FC), in part from the bile, accumulates in the ER membrane of LDLR deficient mice. In contrast, LCAT/LDLR deficient mice have reduced levels of ER membrane FC and are resistant to cholesterol diet-induced elevations, in part from increased INSIG-1 expression and cholesterol esterification by ACAT2. Our analysis has led to the first report of cholesterol-induced hepatic ER stress in vivo and the identification of ER FC levels as a critical indicator of ER stress susceptibility.

Research Doctorate - Doctor of Philosophy (PhD) / Melanoma is a skin cancer that remains a major public health problem in Australia because of its high incidence and the high morbidity and mortality associated with the disease. Melanoma has proven largely resistant to many chemotherapeutic and biological agents. Hope for a new approach in treatment of melanoma comes from the identification of the mechanisms employed in induction of apoptosis by ER stress and the possible resistance mechanisms in melanoma cells against ER stress-induced apoptosis. At the beginning of this study, little was known about the effects of ER stress on melanoma. The aim of this thesis was to elucidate the mechanisms of ER stress-induced apoptosis, the interaction between ER stress pathways and other signalling pathways in melanoma, thus to provide more information in identification of treatment approaches that will increase the sensitivity of melanoma to apoptosis induced by ER stress. Studies in Chapter 3 show that most melanoma cells are relatively resistant to ER stress-induced apoptosis except one cell line Me1007. However, inhibition of the MEK/ERK sensitizes melanoma cells to ER stress-induced apoptosis. This is mediated, at least in part, by caspase-4 activation and is associated with inhibition of the ER chaperone GRP78 expression. Moreover, inhibition of the MEK/ERK pathway reduces the level of GRP78 expression as well as its up-regulation by ER stress. Therefore, when the MEK/ERK is inhibited, caspase-4 is released from its complex with GRP78 and activated to mediated apoptosis. Chapter 4 demonstrates that up-regulation of the anti-apoptotic Bcl-2 family member Mcl-1 is one of the mechanisms critical for protection of melanoma cells against ER stress-induced apoptosis. Inhibition of Mcl-1 by siRNA renders melanoma cells sensitive to apoptosis induced by the ER stress inducers Thapsigargin (TG) or Tunicamycin (TM) mediated by PUMA and Noxa. ER stress up-regulates the BH3-only proteins PUMA and Noxa, but not Bim and BIK in melanoma cells, through transcriptional mechanisms, but the increase of Noxa but not PUMA is dependent on p53. Up-regulation of Mcl-1 is also due to increased transcription that involved the IRE1α and ATF6 signaling pathways of the unfolded protein response. In addition, activation of the MEK/ERK signaling pathway appears to be necessary for optimal up-regulation of Mcl-1. Melanoma cells are largely unresponsive to chemotherapy-induced apoptosis. Activation of the Unfolded Protein Response (UPR) by ER stress has profound effects on the sensitivity of melanoma cells to clinically relevant chemotherapeutic drugs and those in development for clinical use. In Chapter 5, the DNA-damaging drugs Cisplatin and Adriamycin, and the histone deacetylase inhibitors Suberic Bishydroxamate (SBHA) and Sodium Butyrate (NaB) further activate the UPR, indicative of induction of ER stress. The MEK inhibitors U0126 and AZD6244 reduce GRP78 expression levels; however, microtubule-targeting drugs Vincristine and Docetaxel do not change the GRP78 level. Knockdown of the IREα and ATF6 pathway of the UPR, and GRP78 by siRNA results in increased sensitivity of melanoma cells to these compounds. Studies in Chapter 6 show that treatment with either Tunicamycin (TM) or Thapsigargin (TG) selectively up-regulates TRAIL-R2 expression and enhances TRAIL-induced apoptosis in melanoma cells. This appears to be cooperatively mediated by the ATF6 and IRE1α signaling pathways and GADD153/CHOP. However, although siRNA knockdown of ATF6 or IRE1α inhibits up-regulation of TRAIL-R2, it sensitizes melanoma cells to TRAIL-induced apoptosis. Thus, it appears that regulation of TRAIL-R2 expression is not the only means by which the UPR regulates TRAIL-induced apoptosis in melanoma. The UPR may also antagonize TRAIL-induced apoptotic signaling by an intracellular mechanism(s). Study of a melanoma cell line Me1007 in Chapter 7 is the only cell line sensitive to ER stress-induced apoptosis, shows that apoptosis in this cell line is induced by ER stress via a caspase-8-mediated pathway. The high sensitivity of Me1007 to ER stress-induced apoptosis is associated with low expression levels of the apoptosis repressor with caspase recruitment domain (ARC) protein. In resistant cell lines, ARC is expressed at relatively high levels, which may effectively inhibit activation of caspase 8. Therefore, ARC appears to be critical in blocking activation of casapse-8 in melanoma cells subjected to ER stress.

The identification of Mouse Mammary Tumour Virus (MMTV) as the causal factor for breast cancer in mice, initiated investigation into a viral cause for human breast cancer. MMTV-like virus has been detected in human breast cancers, lymphomas and primary biliary cirrhosis (PBC), suggesting the virus is not restricted to human breast cancers. We hypothesized that the virus is detected in human tissues influenced by steroid hormones. We detected a region of the envelope (env) gene of MMTV-like virus in 53/210 (25%) of liver disease, 4/21 (19%) of hepatocellular carcinoma (HCC), 14/89 (16%) of ovarian cancer, 53/147 (36%) of prostate cancer, 5/50 (10%) of endometrial cancer and 13/141 (9%) of skin cancer samples but not in lung cancers (0/51). Viral env DNA was also detected in 4/81 (5%) of placentae and 5/90 (6%) of breast milk cells from healthy women whilst viral env RNA was detected in 2/90 (2%) of breast milk supernatants and (0/81) placentae. Immunohistochemistry staining for the presence of estrogen receptor alpha (ER-??) and progesterone receptor (PgR) demonstrated a significant association between ER-??/PgR and MMTV-like virus in human ovarian, prostate, endometrial and skin cancers though no significant association was observed between ER-??/PgR and the virus in liver tissues. We were also unable to demonstrate a significant association between accumulation of p53 tumour suppressor protein and MMTV-like virus in liver disease and HCC. Despite the demonstration of viral env integration in genomic DNA from human placentae using Southern Blots, other regions of the virus were not detected following PCR attempts with published primer sets. This study adds to the current knowledge of distribution of MMTV-like virus in humans. The detection of the virus in hormonally influenced human tissues (positive for ER-?? or PgR) indicates an association between MMTV-like virus and steroid hormones in some human tissues. The detection of the virus in placentae and breast milk also suggests potential routes of transmission of the virus in humans. Although the exact role of the virus in these tissues is not known, the presence of the virus together with other genetic alterations and/or the influence of steroid hormones could be involved in the transformation of various human tissues (i.e.pathogenesis).

The functional integrity of the proteome is essential for proper cell functioning. Protein homeostasis, or proteostasis, is maintained by a network of pathways that mediate the biosynthesis, folding and degradation of proteins. Accumulating evidence suggest that ageing is associated with a general decline in protein homeostasis. Proteins are synthesised in the cytosol as extended polypeptide chains, which must then be folded in to their native conformation, before moving to their site of function. Protein folding occurs in different subcellular compartments, such as the cytosol and the endoplasmic reticulum (ER). Due to the nature of the proteins that are processed in the ER (e.g. ionic channels, receptors, hormones, signalling molecules), the function of this compartment can be seen to be of vital importance to the cells. However, the effect of age on ER protein homeostasis is virtually unknown. A combination of post-mortem mouse tissues and cell-based models were used to examine the impact of age on ER protein folding, quality control and the ER stress response. Analysis of the expression level of ER-resident and ER-linked proteins showed a number of age-related changes in mouse tissues, in human fibroblasts aged in vitro by serial passage, and human fibroblasts obtained from young and old donors (in vivo aged cells). Overall, the pattern of changes was variable between different tissues and cell systems. However, a common feature of aged tissues and both cellular models of ageing, was a significant increase in phosphorylation of eIF2alpha, indicating that ER protein homeostasis is affected with age. In addition, ageing in several tissues and in both cellular systems was associated with accumulation of polyubiquitinated substrates, suggesting that degradation of abnormal proteins via the proteasome is deficient. More detailed investigation of ER proteostasis using the cellular models showed that in vitro aged cells had a decreased capacity to fold a temperature sensitive model membrane protein (ts O45 VGV-G) and were less efficient at degrading two model substrates of the ER-associated degradation pathway. Since the ER protein folding and degradation pathways are modulated by ER stress response signalling to restore ER homeostasis, therefore their malfunctioning would impact on the ability of aged cells to cope with stress. Indeed, in the aged cells both PERK and IRE-1 signalling were perturbed. In addition, the level of BiP was not upregulated following ER stress in the aged cells. Moreover, ER stress induction led to decreased cell survival in the case of aged cells, showing that the inability of aged cells to maintain ER protein homeostasis results in increased susceptibility to cell death. All these data together shows how perturbed ER proteostasis can occur with increased age and its impact on overall cell wellbeing, therefore provide new insight into mechanisms of cell ageing.

MPEG compressed video is very sensitive to data loss; not only may corruption caused by the loss of an ATM cell be quite severe, but the use of inter-frame predictive coding may cause the corruption to persist or even spread over a period of several frames. In order to lessen the impact of cell loss on compressed video, several people (for example; Ghanbari, Kishino et al.) have proposed techniques for splitting the coded bitstream into two layers of differing importance to the decoded video quality, and transmitting these layer at different cell loss priorities by utilising the ATM cell loss priority bit. The great improvements gained from the use of such layered coding and transmission schemes raise the question of how much further improvement might be gained if we had more ATM cell priority levels to use. This thesis presents an investigation into the performance benefits of MPEG video transmission over a hypothetical ATM network supporting four, as opposed to the standard two, priority levels. The investigation considers three prioritisation schemes: a simple un-layered method that prioritises cells based upon the estimated level or corruption within the decoded video that may occur as a result of losing each cell, a two layer coding scheme based upon the MPEG-2 data partitioning method, and an extension of the two layer scheme to use three layers. The performance for each of these coding and prioritisation methods is evaluated in terms of the decoded root mean square error (RMSE), by transmission over a simulated one, two and four priority level ATM network. The results appear to indicate that while prioritisation of an un-layered MPEG video cell stream using two priority levels is of some benefit, extending the scheme to utilise four priority levels is of only limited effectiveness. The layered coding results meanwhile show that the new low priority layers created by extending data partitioning beyond two layer coding are not of significantly different importance to the decoded RMSE video quality to warrant transmission at different priorities, and that for the systems tested, four priority levels would again offer only a modest improvement in performance over two priority levels. Subjective video quality evaluation tests however confirm a definite difference in subjective importance between the layers, and suggest a performance improvement for four priority level transmission.