Browsing by Subject "Hsp90"
Results Per Page
Sort Options
Item Open Access Extracellular Hsp90 is Actively Trafficked and Internalized in Breast Cancer Cells(2016) Crowe, Lauren BurianekDespite its ubiquitous abundance, Hsp90 inhibitors have shown promise in anti-cancer clinical trials, suggesting that Hsp90 inhibitors selectively target tumor cells while exhibiting minimal effects in normal cells. Extracellular expression of heat shock protein 90 (eHsp90) by tumor cells is strongly correlated with malignancy. Development of small molecule probes that can specifically detect eHsp90 in vivo may therefore have utility in the early detection of malignancy. We synthesized a fluorescent cell impermeable Hsp90 inhibitor, HS-131, to target eHsp90 in vivo. HS-131 was characterized biochemically to ensure specificity for eHsp90, and an inactive analog was also synthesized to be used as an in vivo control.
Through confocal microscopy, eHsp90 can be visualized with cell impermeable, fluorophore-tagged Hsp90 inhibitors. High resolution confocal and real time lattice light sheet microscopy showed that probe-bound eHsp90 accumulates in punctate structures on the plasma membrane of breast tumor cells and is subsequently actively internalized. This internalization occurs in the presence and absence of inhibitors. The extent of internalization correlates with tumor cell aggressiveness, and this process can be induced in benign cells by over-expressing p110HER2, leading to malignant transformation of these cells. Internalization of eHsp90 is also increased after inhibition of Hsp70, suggesting that overcompensation of the heat shock response can also upregulate the eHsp90 trafficking mechanism. Whole body 3D cryo fluorescence imaging and histology of flank and spontaneous tumor-bearing mice strongly suggests that eHsp90 expression is a unique phenomenon in vivo.
Taken together, these results suggest that active and differential internalization of eHsp90 in aggressive cancer cells contributes to the selectivity observed upon Hsp90 inhibitor treatment and may provide a novel metastatic biomarker for solid tumors and may lead to the development of a tumor-specific drug delivery system.
Item Open Access Hs-27, a Novel Hsp90 Inhibitor, Exhibits Diagnostic and Therapeutic Potential in Triple Negative Breast Cancer(2016-04-22) Belonwu, StellaHeat-shock protein 90 (Hsp90) is a molecular chaperone that is ubiquitously expressed in all cell types and essential for maintaining cell homeostasis by assisting in protein folding, de-aggregation, and degradation. Hsp90 is upregulated in all breast tumors, where it is present on the cell surface, unlike in normal cells, and supports signal transduction pathways important for tumor progression. Hence, Hsp90 has emerged as an attractive anti-cancer target. Triple negative breast cancer (TNBC) is a highly aggressive and difficult to treat subtype of breast cancer. Because TNBC is unresponsive to hormone therapies, there are no good therapy options available. Thus, Hsp90 may serve as a reasonable target for TNBC. Hs-27 is a novel Hsp90 inhibitor made by Dr. Timothy Haystead of Duke University’s Department of Pharmacology and Cancer Biology. It was developed with a fluorescein contrast agent, which makes it suitable for diagnostics. Preliminary experiments with Hs-27 with breast cancer cell lines of different receptor subtypes show that it binds to ectopically expressed Hsp90 in tumor cells. In vitro therapy experiments also show that Hs-27 down-regulates client proteins implicated in tumor growth. In this study, I further establish Hs-27’s diagnostic and therapeutic ability in vivo through hyperspectral and fluorescence imaging in dorsal skinfold window chamber tumor models in mice. Largely, I observed that at lower doses, Hs-27 allows for real-time, non-invasive imaging for cancer detection and at higher doses has the potential for therapeutic benefits.Item Open Access Identification of Essential Functions of GRP94 in Metazoan Growth Control and Epithelial Homeostasis(2009) Maynard, Jason ChristopherGRP94, the endoplasmic reticulum Hsp90, is a metazoan-restricted chaperone essential for early development in mammals, yet dispensable for mammalian cell viability. These data suggest that GRP94 is required for important developmental processes relying on cell-cell communication and cell-cell interaction. Consistent with this hypothesis, loss of GRP94 expression in mouse is embryonic lethal yet tissue culture cells expressing no GRP94 are viable. To date, functional studies of GRP94 have relied on cell-autonomous model systems, the use of which has lead to discoveries of proteins that GRP94 chaperones also called client proteins. These systems give limited insight into the essential role(s) played by GRP94 in metazoan biology. The dichotomy that GRP94 is necessary for metazoan life, but dispensable for cellular viability suggests that the chaperone is required for the functional expression of secretory and/or membrane proteins that enable cells to function in the context of tissues.
To explore this hypothesis, the Drosophila ortholog of GRP94, Gp93, was identified and Gp93 mutants were created using imprecise P-element excision. Gp93 was found to be an essential gene in Drosophila. Loss of zygotic Gp93 expression is late larval lethal and causes prominent defects in the larval midgut, the sole endoderm-derived larval tissue. Gp93 mutant larvae display pronounced defects in the midgut epithelium, with aberrant copper cell structure, markedly reduced gut acidification, atypical septate junction structure, depressed gut motility, and deficits in intestinal nutrient uptake. The metabolic consequences of the loss of Gp93-expression are profound; Gp93 mutant larvae exhibit a starvation-like metabolic phenotype, including suppression of insulin signaling and extensive mobilization of amino acids and triglycerides. The defects in copper cell structure/function accompanying loss of Gp93 expression resemble those reported for mutations in labial, an endodermal homeotic gene required for copper cell specification, and α-spectrin, thus suggesting an essential role for Gp93 in the functional expression of secretory/integral membrane protein-encoding labial protein target genes and/or integral membrane protein(s) that interact with the spectrin cytoskeleton.
The creation of Gp93 mutant Drosophila has allowed for the study of GRP94 function in vivo and will be of upmost importance to future studies examining the function of this chaperone in all aspects of metazoan biology. This dissertation focuses on the morphological and physiological defects that accompany loss of Gp93 expression in Drosophila larvae. It will also outline future studies utilizing this model.
Item Embargo Investigation of Heat Shock Protein 90 in Plasmodium Parasites(2024) Mansfield, Christopher RockyMalaria is an infectious disease caused by apicomplexan Plasmodium species. These protozoan parasites are transmitted by a mosquito vector to a human host, wherein they undergo an asymptomatic liver stage followed by a symptomatic blood stage of infection. Despite eradication efforts, Plasmodium remain accountable for hundreds of thousands of mortalities per year, mostly caused by P. falciparum. The spreading resistance to the front-line antimalarials that broadly disrupt parasite proteostasis demands further characterization of their adaptive stress responses and novel multi-stage drug targets. This work focuses on the essential P. falciparum molecular chaperone heat shock protein 90 (PfHsp90). Specifically, PfHsp90 is expected to directly interact with a subset of parasite proteins to facilitate their ATP-dependent maturation, stabilization, and regulation. Despite this critical function, the scope of its chaperoning interactions—as well as its consequent contributions to mitigate cellular stress and maintain parasite proteome integrity throughout development—remains largely unresolved. To enable its functional interrogation, we first aimed to establish chemical inhibitors of PfHsp90 with greater affinity to the parasite compared to the conserved human homolog (HsHsp90). In general, our testing supports the particular utility of compounds that bind at the chaperone’s nucleotide-binding domain, as opposed to a putative C-terminal allosteric site, based on their high affinity and resolved mode of ATP-competitive inhibition. From this class of competitive inhibitors, we identified XL888 as exhibiting moderate selectivity to PfHsp90, despite that it was initially developed as a HsHsp90 inhibitor. Subsequent structural evaluation indicated that the PfHsp90 lid subdomain contributes to the parasite chaperone’s higher affinity interaction with XL888’s tropane scaffold. Considering this molecular basis, we were able to develop Tropane 1 as a novel XL888 analog with nanomolar affinity and approximately 10-fold selectivity to PfHsp90, which further demonstrated dual-stage, anti-Plasmodium activity. We next surveyed the PfHsp90-dependent proteome using innovative chemical biology strategies. Based on their thermal stability after chaperone inhibition, we identified 50 candidates as putative PfHsp90 interactors. A significant enrichment of proteasome regulatory particle components was represented in this analysis, from which we subsequently validated that PfHsp90 chaperones the 26S proteasome to support the controlled recycling of cellular proteins. Additionally, we adopted bio-orthogonal labeling with unnatural amino acids to track proteome dynamics in Plasmodium parasites. To date, we have implemented this approach to support that compromised PfHsp90 activity coordinates translation attenuation as a stress response. However, this work sets the foundation to employ such labeling to quantify PfHsp90-coordinated proteome dynamics across multiple parasite life stages. Collectively, these findings broaden our understanding of PfHsp90’s regulation of Plasmodium parasite proteostasis and further establish the potential of this molecular chaperone as a novel, multi-stage antimalarial drug target.
Item Open Access Leveraging Tumor Stress Responses for a See and Treat Paradigm in Breast Cancer: Applications in Local and Global Health(2018) Crouch, Brian ThomasWith the widespread adoption of mammograms for early breast cancer detection in high income countries (HICs), modern research has principally pivoted towards a focus on reducing overtreatment of patients, particularly those with early stage breast cancer. There are numerous examples of clinical practices and regulations that reflect this shift, including changes in screening recommendations, patient monitoring, re-excision guidelines, and genetic testing, all of which seek to reduce unnecessary intervention without compromising patient outcomes.
Despite changing guidelines, there remains a distinct lack of technologies to reduce overtreatment while ensuring the best possible outcome for patients. One such example is Breast Conserving Surgery (BCS) followed by radiation therapy. There is a wide-range of re-excision rates reported in the literature, but most groups report that 20-40% of patients undergo at least one re-excision. Taking additional shavings during BCS, new guidelines dictating relationships between margin status after BCS and re-excision, and radiation therapy all strive to maximize removal of residual tumor cells with as few surgeries as possible in patients with a new breast cancer diagnosis. However, secondary cancers from radiation therapy, the potential for cancer dissemination as a result of re-excision surgeries, and the burgeoning costs of repeat visits and interventions to an already depleted health care system necessitate new and innovative solutions to improve health outcomes while reducing health expenditures.
While seeking to improve patient experience in HICs, many women in low to middle income countries (LMICs) are unable to access adequate screening and life-saving treatments. Even those who manage to receive a proper diagnostic test are all too frequently lost to follow up care, leading to a disproportionately high burden of breast cancer mortality in LMICs.
The goal of the work presented here is to reduce overtreatment in HICs while simultaneously minimizing access barriers to screening and treatment for women in LMICs through developing a rapid and low-cost molecular diagnostic platform for breast cancer. In HICs, this diagnostic platform could be deployed at two points in the breast cancer care cascade: 1) during diagnostic biopsy to ensure adequate lesion sampling at the point-of-diagnosis, and 2) during intraoperative margin assessment as a ‘see-and-treat’ paradigm that utilizes a single agent to guide surgical resection and to treat residual disease during surgery. In LMICs where limited access to tissue processing equipment and a pathologist often render histological examination of tissue impossible, the diagnostic platform could be used to cheaply and robustly diagnose tissue at the point-of-care.
Three specific aims were proposed to develop the diagnostic platform. The first aim was to demonstrate a single-agent see-and-treat paradigm in pre-clinical models of breast cancer using a fluorescent tracer across all subtypes of breast cancer. The diagnostic piece began by showing that a fluorescently-tethered Hsp90 inhibitor (HS-27), made up of an Hsp90 inhibitor previously used in clinical trials tethered to a fluorescein isothiocyanate (FITC) derivative, is taken up by breast cancer cells in vitro regardless of receptor subtype, and that blocking the ATP binding pocket of Hsp90 leads to reduced HS-27 fluorescence, confirming that fluorescence is a result of HS-27 bound to its target. The in vitro study was expanded to define the therapeutic potential of HS-27 by demonstrating degradation of Hsp90 client proteins consistent with Hsp90 inhibition, and reduction of cellular metabolism, confirming protein degradation led to downstream effects on its signaling pathway. To round out the therapy component of our ‘see-and-treat’ paradigm, HS-27 treatment was found to reduce cell proliferation rates across breast cancer receptor subtypes.
The diagnostic component was moved into animals using a dorsal skinfold window chamber model to interrogate HS-27 uptake in vivo in the context of tumors and their surrounding microenvironment. As expected, HS-27 uptake was significantly greater in tumor window chambers than in non-tumor controls. Utilizing a fluorescent glucose analog to examine glucose uptake levels in tumors as a surrogate for aggressive disease showed that HS-27 strongly correlated (R2 = 0.96) with glucose uptake, suggesting surface Hsp90 expression is upregulated in aggressive glycolytic tumors.
To finish aim 1, HS-27 staining was performed on tumors ex vivo, achieving comparable contrast to in vivo agent administration, providing a path towards translating HS-27 to ex vivo clinical use. A small ex vivo pilot clinical study in patients undergoing diagnostic biopsy revealed a significant correlation between HS-27 uptake and the percentage of tumor present in the sample, providing first proof-of-principle of our HS-27 fluorescence-based diagnostic platform in patients. HS-27 was first imaged in biopsies in order to enroll patients across different receptor subtypes rather than at surgery where the majority of patients have estrogen receptor positive (ER+) disease. To summarize, aim 1 demonstrated a ‘see-and-treat’ paradigm in pre-clinical models of breast cancer, and provided a path towards moving HS-27 into the clinic.
With proof-of-principle patient results revealing that HS-27 may be a feasible diagnostic tool, the focus of aim 2 transitioned towards optimizing the imaging system and protocol. The ex vivo imaging strategy was optimized to minimize non-specific HS-27 uptake in preclinical models. Imaging parameters were fully vetted in a clinical study designed to interrogate HS-27 uptake in patients with breast cancer or benign conditions, as well as in a disease-free population. A high-resolution microendoscope (HRME) designed to image FITC fluorescence in a pre-clinical biopsy model was used to investigate how time between tissue excision and imaging, agent incubation time, and agent dose affect the specificity of HS-27 based diagnostics. For these experiments, a modified version of HS-27 with a 100-fold reduction in Hsp90 affinity, called HS-217, was used to establish non-specific fluorophore uptake. Calculating the ratio of HS-27 fluorescence to HS-217 fluorescence provided a ‘specificity ratio’ that was maximized with a post-excision window up to 10-minutes, 1-minute incubation time, and 100 µM dose.
The optimized protocol was then tested in 37 patients undergoing ultrasound-guided core needle biopsy and in 6 disease-free patients undergoing breast reduction mammoplasty. HS-27 uptake was significantly greater in tumor samples than mammoplasty control samples. Interestingly, HS-27 uptake was similar in tumor and benign lesion samples on average, however, examining the distribution of fluorescence across the biopsy reveals different staining patterns between tumor and benign lesions. Concurrent with the finding in aim 1 that HS-27 levels are elevated in aggressive tumors, HS-27 strongly and inversely correlated with the presence of tumor infiltrating lymphocytes, a positive prognostic marker in Her2+ and triple negative breast cancers. Additionally, leveraging both intensity and spatial patterns to generate a Gaussian support vector machine classifier allowed for accurate classification of tumor, benign lesion, and mammoplasty samples. Classification of tumor vs benign lesions resulted in an area under the receiver operating characteristic curve (AUC) of 0.93 with a sensitivity of 82% and specificity of 100%. Classification of tumor vs mammoplasty samples resulted in an AUC of 0.96 with a sensitivity of 86% and specificity of 100%.
So far, HS-27 uptake has been shown to be specific to tumor over non-tumor tissues, increased HS-27 fluorescence was suggestive of an aggressive tumor phenotype, and ex vivo HS-27 imaging accurately distinguished tumor from both benign and normal breast tissue. Two limitations of the imaging system used in aims 1 and 2 were: 1) the requirement to place the HRME probe in contact with the tissue, potentially causing artificial changes in signal due to pressure differences during probe placement, and 2) the small field of view, which prohibited translation to samples larger than 1-2 cm. Thus, the goal of aim 3 was to develop a wide-field, non-contact imaging system to demonstrate feasibility of translating ex vivo HS-27 imaging to multiple points in the breast cancer care cascade.
We have previously developed a Pocket colposcope for cervical pre-cancer detection and have recently completed construction and testing of an alpha prototype. The colposcope contains a 5 MP camera and white and green light emitting diodes (LEDs) on the tip. It weighs 1 pound, and interfaces with a phone, tablet, or computer, which provides power to the device and enables image capture. The Pocket colposcope, which will now be referred to as a Pocket mammascope is well-suited for breast margin imaging with the ability to, survey breast tumor margins as large as 10 -cm2 in a few snapshots, while maintaining the ability to image a cluster of tumor cells on a length scale of several microns.
The Pocket colposcope was modified into a Pocket mammoscope to perform fluorescence imaging through the addition of a collar with excitation LEDs and a bandpass filter for fluorescence collection. A series of bench tests show that the Pocket mammoscope can perform fluorescence imaging in a wide-field mode with a diagonal field of view of 3.25 cm (compared to 750 µm with the HRME) at a resolution of 25 µm (compared to ~4 µm with the HRME), and high-resolution mode with a diagonal field of view of 1.25 cm and resolution of 12 µm. The two imaging modes are easily navigated between through the use of a simple slider mechanism. The Pocket mammoscope was next used to image HS-27 fluorescence across in vivo and ex vivo models, with comparable results to our previous imaging systems. Additionally, the optimized ex vivo imaging protocol from aim 2 was used to shown to be compatible with the Pocket mammoscope in a cohort of patients undergoing standard-of-care ultrasound-guided core needle biopsy, and that that Pocket mammoscope is capable of imaging an entire biopsy in a single snapshot. Proof-of-concept translation to intraoperative margin assessment utilizing a window chamber model, similar to aim 1, validated that the Pocket mammoscope could image HS-27 both systemically and topically delivered to a tumor.
In conclusion, this work set out to provide a theranostic tool to reduce overtreatment for patients with breast cancer in HICs, and provide a rapid diagnostic test implementable at the point-of-care in LMICs. Towards these goals, aim 1 showed that HS-27 uptake is higher in more aggressive tumors, potentially serving as a prognostic marker delineating which patients require more or less aggressive treatment regimens. Aim 2 found that a Gaussian support vector machine classification scheme based on features from ex vivo HS-27 images accurately distinguishes tumor from both benign conditions and normal breast tissue. Finally, aim 3 demonstrated the feasibility of translating HS-27 to both diagnostic biopsy and intraoperative margin assessment by creating a Pocket mammoscope capable of imaging an entire biopsy and a tumor margin in a few snapshots. Ultimately, this work demonstrates that HS-27 imaging with the Pocket mammoscope is a means for rapid, automated detection of breast cancer, regardless of subtype, which could improve breast cancer management in both HICs and LMICs.
Item Open Access Regulation of the Apoptosome in Cancer(2012) Kim, JiyeonApoptosis is a cellular suicide program that can be initiated by various genotoxic and cytotoxic stimuli. In many cases, such cell damaging agents promote cell death through the intrinsic apoptotic pathway by triggering mitochondrial cytochrome c release and subsequent caspase activation. Cytosolic cytochrome c is directly responsible for initiating formation of the caspase-activating apoptosome, which plays a crucial role in the apoptotic process. Given the importance of cellular fate, apoptosis is tightly controlled by a balance between survival and death signals. It has been shown that activated cell survival pathways, including the mitogen-activated protein kinase (MAPK) cascade and the PI3K/Akt signaling, enhance cell viability by conferring resistance to apoptotic cell death. However, the underlying mechanism(s) that lead to inhibition of functional apoptosome formation (and caspase activation) has yet to be elucidated. In the studies that are described in this dissertation, I have investigated the regulation of apoptosis downstream of mitochondrial cytochrome c release with the goal of understanding how survival signaling can alter the apoptotic program, contributing to human malignancies.
First, we describe a mechanism for the inhibition of cytochrome c-induced caspase activation by MAPK signaling, identifying a novel mode of apoptotic regulation exerted through Apaf-1 phosphorylation by the 90-kDa ribosomal S6 kinase (Rsk). We have found that recruitment of 14-3-3ε to phosphorylated Ser268 impedes the ability of cytochrome c to nucleate apoptosome formation and activate downstream caspases. High endogenous levels of Rsk in PC3 prostate cancer cells or Rsk activation in other cell types promoted 14-3-3ε binding to Apaf-1 and rendered the cells insensitive to cytochrome c, suggesting a role for Rsk signaling in apoptotic resistance of prostate cancers and other cancers with elevated Rsk activity. These results identify a novel locus of apoptosomal regulation wherein MAPK signaling promotes Rsk-catalyzed Apaf-1 phosphorylation and consequent binding of 14-3-3ε, resulting in decreased cellular responsiveness to cytochrome c.
In the second part, we examine how apoptosis is inhibited by oncogenic tyrosine kinase signaling by using leukemogenic tyrosine kinase-induced leukemia model systems. We have demonstrated that protein phosphatase 5 (PP5) is responsible for Hsp90β hypophosphorylation, which can contribute to impaired cell death in leukemia expressing oncogenic tyrosine kinases. Loss of PP5 results in an increase of Hsp90β phosphorylation, raising leukemic cells' responsiveness to imatinib, a BCR-ABL kinase inhibitor. Further we have discovered that acetylation regulates PP5 activity on Hsp90β. Mutational study showed that K144 acetylation on PP5, which was diminished in leukemic conditions, inhibited PP5 binding to Hsp90β, causing Hsp90β hyperphosphorylation and subsequently potentiating cells to apoptosis. These studies reveal a molecular mechanism by which agents enhancing PP5 acetylation may be a potential treatment for leukemias. Collectively, this work provides new insight into mechanisms of regulation of apoptosome formation/function, helping us understand how the evasion of apoptotic cell death contributes to cancer cell survival. Further, this finding implicates cytochrome c-induced apoptotic signaling in the context of cancer cell responsiveness to chemotherapeutic treatments.
Item Open Access Targeting Borrelia burgdorferi's Heat Shock Protein for the Diagnosis and Treatment of Lyme Disease(2020) Sell, MadelineInfections are most commonly identified by microscopy, culturing the organism, or testing the patients blood for antigens or antibodies. These methods are unreliable in bacteria that persist in a non-dividing, metabolically inactive dormant state, leading to treatment delays and an increased risk of developing chronic morbidities. Borrelia burgdorferi (B. burgdorferi), the causative spirochete in Lyme Borreliosis, is an example of a stealth pathogen difficult to culture from blood, capable of evading the host immune system, and under adverse growth conditions in host tissue, can survive in a dormant state. Despite early diagnosis and treatment, 20-35% of patients with Lyme Borreliosis experience chronic symptoms, the etiology of which remains unknown due to the lack of accurate diagnostics to demonstrate the presence of a persistent infection. In vivo diagnostic imaging of bacterial infections is currently reliant on targeting their metabolic pathways, an ineffective method to identify microbial species with low metabolic activity. Here we characterize HS-198 as a small molecule-fluorescent conjugate that selectively targets the highly conserved bacterial protein, HtpG (High temperature protein G) within B. burgdorferi, the bacteria responsible for Lyme Disease. We describe the use of HS-198 to target morphologic forms of B. burgdorferi in both the logarithmic growth phase and the metabolically dormant stationary phase. Furthermore, in a murine infection model, systemically injected HS-198 identified B. burgdorferi as revealed by imaging in post necropsy tissue sections. These findings demonstrate how small molecule probes directed at conserved bacterial protein targets can function to identify the microbe using non-invasive imaging and potentially as scaffolds to deliver antimicrobial agents to the pathogen, potentially solving both the problem of diagnosis and treatment.