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Fibronectin is localized to late endosome compartments which require intact tubulin microtubules to traffic to the plasma membrane. (A) To examine inhibitor effects on secretion of FN1, an ELISA assay was performed on tissue culture media (HPC-1) collected from LNCaP cells treated with AUY922 (25 nM) for 48 h and controlled for cell number. Date represents mean ± SEM of 4 independent experiments. (B) Model illustrating cytosolic vesicle and compartment trafficking to the plasma membrane along microtubule networks. (C) Co-location of FN1 with vesicle markers including endosomes (EEA1, Appl1, Rab5, Rab7), lysosome (LIMP2, LAMP1), endoplasmic reticulum (PDI) and Golgi (Tgn46) was examined by fluorescence microscopy in LNCaP cells treated with DMSO for 48 h and probed for vesicle markers (red) and FN-1 (green) and DAPI mount media (blue). Images were taken using Leica SP8 confocal microscope at 63x magnification.
Source publication
The molecular chaperone Hsp90 is overexpressed in prostate cancer (PCa) and is responsible for the folding, stabilization and maturation of multiple oncoproteins, which are implicated in PCa progression. Compared to first-in-class Hsp90 inhibitors such as 17-allylamino-demethoxygeldanamycin (17-AAG) that were clinically ineffective, second generati...
Contexts in source publication
Context 1
... cell and subsequent incorporation into the ECM 20,21 . However, despite the marked induction of FN1 expression in AUY922 treated PCa cells, ELISA assays performed to detect FN1 in the culture medium demonstrated that AUY922 (25 nM) caused a consistent and significant reduction in FN1 secretion by LNCaP cells compared to vehicle treated cells ( Fig. 4A; n = 4 independent ...
Context 2
... cellular secretion of proteins is reliant on endosome traffic along microtubules for delivery to the plasma membrane, where the release of ECM proteins including FN1 occurs, allowing for their polymerization and deposition in the extracellular space (Fig. 4B). To determine which cytosolic compartments contained FN1, co-staining of FN1 was performed in LNCaP cells with a panel of endoplasmic reticulum, Golgi, endosome and lysosome vesicle markers including PDI, Tgn46, Appl1, EEA1, Rab5, Rab7, LIMP2 and LAMP1 (Fig. 4C). FN1 co-located with PDI (endoplasmic reticulum), Tgn46 (Golgi) and, most ...
Context 3
... occurs, allowing for their polymerization and deposition in the extracellular space (Fig. 4B). To determine which cytosolic compartments contained FN1, co-staining of FN1 was performed in LNCaP cells with a panel of endoplasmic reticulum, Golgi, endosome and lysosome vesicle markers including PDI, Tgn46, Appl1, EEA1, Rab5, Rab7, LIMP2 and LAMP1 (Fig. 4C). FN1 co-located with PDI (endoplasmic reticulum), Tgn46 (Golgi) and, most robustly, vesicles containing the Rab5 (early endosomal) and Rab7 (late endosomal) markers (Fig. 4C). Visual inspection suggests that colocation of FN1 with these markers was unaltered by AUY922 (Supplementary Figure ...
Context 4
... performed in LNCaP cells with a panel of endoplasmic reticulum, Golgi, endosome and lysosome vesicle markers including PDI, Tgn46, Appl1, EEA1, Rab5, Rab7, LIMP2 and LAMP1 (Fig. 4C). FN1 co-located with PDI (endoplasmic reticulum), Tgn46 (Golgi) and, most robustly, vesicles containing the Rab5 (early endosomal) and Rab7 (late endosomal) markers (Fig. 4C). Visual inspection suggests that colocation of FN1 with these markers was unaltered by AUY922 (Supplementary Figure ...
Context 5
... alternative hypothesis was that AUY922 may affect FN1 secretion via impaired exosome release, as FN1 is a reported exosomal cargo protein in PCa cell lines and patient serum 22,23 . However, there was no consistent effect of AUY922 on exosome release in 3 independent cell lines (LNCaP, PC-3 and 22Rv1) (Supplementary Figure 4), and the FN1 content of those exosomes was only modestly reduced by AUY922 (Supplementary Figure 5). Depletion of cellular fibronectin suppresses PCa cell invasion to a similar extent as Hsp90 inhi- bition. ...
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Unconventional prefoldin RPB5 interactor (URI, or RMP, a member of the prefoldin family of molecular chaperones) exhibits oncogenic activity in several types of cancer, including ovarian cancer. However, the underlying regulatory mechanism in ovarian cancer remains unclear. MicroRNAs (miRNAs) negatively regulate gene expression, and their dysregula...
Citations
... eHsp90's role in cancer cell invasion has been studied in vitro using Matrigel [3,5,9,10,13,19,31] which mimics the composition of the basement membrane (BM) [32,33]. The BM, primarily composed of Collagen-IV and elastin, is a form of ECM protein that resembles a sheet-like structure and outlines the epithelial cell compartment, separating it from interstitial connective tissue [32,33]. ...
Simple Summary
Breast cancer cells secrete Hsp90, a protein that, inside of cells, regulates the function of hundreds of proteins, but outside of cells, extracellular Hsp90 (eHsp90) can activate a subset of proteins that promote invasion, the first step of metastasis. Blocking eHsp90 in mouse models inhibits metastasis, and we sought to understand how this occurs. Prior studies have predominantly focused on eHsp90 in cancer invasion within the immediate vicinity of the primary tumor, specifically its role in invading outside the epithelial compartment. However, eHsp90’s role in cancer invasion across the extended connective tissue after the cells have crossed the boundary of the epithelial compartment remains unknown. We show here that eHsp90 directly binds to and aligns Collagen-1 fibers, a major structural component of connective tissues, which, when aligned, form highways that allow efficient cancer migration. Our study suggests that the Hsp90 dimer, in its open state, binds to Collagen-1 molecules to align the fibers, which results in enhanced breast cancer invasion through the Collagen-1 matrix. Knowing this could help us propose experiments to test eHsp90 inhibitors for therapeutically targeting metastatic breast cancer.
Abstract
Cancer cell-secreted eHsp90 binds and activates proteins in the tumor microenvironment crucial in cancer invasion. Therefore, targeting eHsp90 could inhibit invasion, preventing metastasis—the leading cause of cancer-related mortality. Previous eHsp90 studies have solely focused on its role in cancer invasion through the 2D basement membrane (BM), a form of extracellular matrix (ECM) that lines the epithelial compartment. However, its role in cancer invasion through the 3D Interstitial Matrix (IM), an ECM beyond the BM, remains unexplored. Using a Collagen-1 binding assay and second harmonic generation (SHG) imaging, we demonstrate that eHsp90 directly binds and aligns Collagen-1 fibers, the primary component of IM. Furthermore, we show that eHsp90 enhances Collagen-1 invasion of breast cancer cells in the Transwell assay. Using Hsp90 conformation mutants and inhibitors, we established that the Hsp90 dimer binds to Collagen-1 via its N-domain. We also demonstrated that while Collagen-1 binding and alignment are not influenced by Hsp90’s ATPase activity attributed to the N-domain, its open conformation is crucial for increasing Collagen-1 alignment and promoting breast cancer cell invasion. These findings unveil a novel role for eHsp90 in invasion through the IM and offer valuable mechanistic insights into potential therapeutic approaches for inhibiting Hsp90 to suppress invasion and metastasis.
... Whole-cell lysates were collected in RIPA lysis buffer supplemented with cOmplete ULTRA protease and phosphatase inhibitor (Cell Signalling Technology (CST), Danvers, MA, USA) and immunoblotting was performed as previously described [19]. Primary and secondary antibodies used in this study are enlisted in Supplementary Table 2. ...
Background
Resistance to androgen receptor signalling inhibitors (ARSIs) represents a major clinical challenge in prostate cancer. We previously demonstrated that the ARSI enzalutamide inhibits only a subset of all AR-regulated genes, and hypothesise that the unaffected gene networks represent potential targets for therapeutic intervention. This study identified the hyaluronan-mediated motility receptor (HMMR) as a survival factor in prostate cancer and investigated its potential as a co-target for overcoming resistance to ARSIs.
Methods
RNA-seq, RT-qPCR and Western Blot were used to evaluate the regulation of HMMR by AR and ARSIs. HMMR inhibition was achieved via siRNA knockdown or pharmacological inhibition using 4-methylumbelliferone (4-MU) in prostate cancer cell lines, a mouse xenograft model and patient-derived explants (PDEs).
Results
HMMR was an AR-regulated factor that was unaffected by ARSIs. Genetic (siRNA) or pharmacological (4-MU) inhibition of HMMR significantly suppressed growth and induced apoptosis in hormone-sensitive and enzalutamide-resistant models of prostate cancer. Mechanistically, 4-MU inhibited AR nuclear translocation, AR protein expression and subsequent downstream AR signalling. 4-MU enhanced the growth-suppressive effects of 3 different ARSIs in vitro and, in combination with enzalutamide, restricted proliferation of prostate cancer cells in vivo and in PDEs.
Conclusion
Co-targeting HMMR and AR represents an effective strategy for improving response to ARSIs.
... Fibrosis is characterized by excessive extracellular matrix (ECM) deposition (including FN), hyperproliferative fibroblasts, and inappropriate matrix remodeling (Wynn and Ramalingam 2012;Li et al. 2018;Henderson et al. 2020), which is linked to high levels of extracellular HSP90 that promote ECM stiffness and correlate with disease severity (Sontake et al. 2017;Bellaye et al. 2018). In the context of cancer, high levels of HSP90 in the tumor microenvironment promote matrix remodeling that stiffens stroma, alters FN proteolytic processing and drives cell invasion and metastasis (Kass et al. 2007, Armstrong et al. 2018, Li et al. 2013Wong and Jay 2016;Baker-Williams et al. 2019). HSP90 inhibition is therefore considered a potential therapeutic strategy to ameliorate the ECM dysregulation characteristic of fibrosis and cancer (Cáceres et al. 2018;Dong et al. 2017;Tomcik et al. 2014;Armstrong et al. 2018). ...
... In the context of cancer, high levels of HSP90 in the tumor microenvironment promote matrix remodeling that stiffens stroma, alters FN proteolytic processing and drives cell invasion and metastasis (Kass et al. 2007, Armstrong et al. 2018, Li et al. 2013Wong and Jay 2016;Baker-Williams et al. 2019). HSP90 inhibition is therefore considered a potential therapeutic strategy to ameliorate the ECM dysregulation characteristic of fibrosis and cancer (Cáceres et al. 2018;Dong et al. 2017;Tomcik et al. 2014;Armstrong et al. 2018). ...
... Exogenous HSP90 increased FN matrix assembly (Hunter et al. 2014;Chakraborty et al. 2020), whereas HSP90β inhibition or knockdown resulted in significant FN matrix turnover and decreased matrix stability in Hs578T cells (Hunter et al. 2014;Boel et al. 2020). In prostate cancer explants, HSP90 inhibitor AUY922 also reduced FN secretion (Armstrong et al. 2018), while FN expression was stimulated by HSP90 inhibitor geldanamycin in an HSF1-dependent manner (Dhanani et al. 2017). Here, to understand better how HSP90 promotes FN matrix assembly, we extend the analysis of the HSP90 -FN interaction and identify critical residues involved in HSP90-mediated FN matrix assembly. ...
HSP90 is a ubiquitously expressed chaperone protein that regulates the maturation of numerous substrate proteins called 'clients'. The glycoprotein fibronectin (FN) is an important protein of the extracellular matrix (ECM) and a client protein of HSP90. FN and HSP90 interact directly, and the FN ECM is regulated by exogenous HSP90 or HSP90 inhibitors. Here, we extend the analysis of the HSP90 - FN interaction. The importance of the N-terminal 70-kDa fragment of fibronectin (FN70) and FN type I repeat was demonstrated by competition for FN binding between HSP90 and the functional upstream domain (FUD) of the Streptococcus pyogenes F1 adhesin protein. Furthermore, His-HSP90α mutations F352A and Y528A (alone and in combination) reduced the association with full-length FN (FN-FL) and FN70 in vitro. Unlike wild type His-HSP90α, these HSP90 mutants did not enhance FN matrix assembly in the Hs578T cell line model when added exogenously. Interestingly, the HSP90 E353A mutation, which did not significantly reduce the HSP90 - FN interaction in vitro, dramatically blocked FN matrix assembly in Hs578T cell-derived matrices. Taken together, these data extend our understanding of the role of HSP90 in FN fibrillogenesis and suggest that promotion of FN ECM assembly by HSP90 is not solely regulated by the affinity of the direct interaction between HSP90 and FN.
... The function of HSP90 and MMP2 interaction is under controlled by tissue inhibitors of metalloproteinase 2 (TIMP2) and ATPase homolog 1 (AHA1) co-chaperones [402]. AUY922, an inhibitor of HSP90, decreases fibronectin secretion into ECM and hampers invasion in prostate cancer [403]. Interestingly, inhibition of HSP90 facilitates decreased contractility and increased TGF-β2 expression of CAFs in prostate cancer [404]. ...
The malignant tumor is a multi-etiological, systemic and complex disease characterized by uncontrolled cell proliferation and distant metastasis. Anticancer treatments including adjuvant therapies and targeted therapies are effective in eliminating cancer cells but in a limited number of patients. Increasing evidence suggests that the extracellular matrix (ECM) plays an important role in tumor development through changes in macromolecule components, degradation enzymes and stiffness. These variations are under the control of cellular components in tumor tissue via the aberrant activation of signaling pathways, the interaction of the ECM components to multiple surface receptors, and mechanical impact. Additionally, the ECM shaped by cancer regulates immune cells which results in an immune suppressive microenvironment and hinders the efficacy of immunotherapies. Thus, the ECM acts as a barrier to protect cancer from treatments and supports tumor progression. Nevertheless, the profound regulatory network of the ECM remodeling hampers the design of individualized antitumor treatment. Here, we elaborate on the composition of the malignant ECM, and discuss the specific mechanisms of the ECM remodeling. Precisely, we highlight the impact of the ECM remodeling on tumor development, including proliferation, anoikis, metastasis, angiogenesis, lymphangiogenesis, and immune escape. Finally, we emphasize ECM "normalization" as a potential strategy for anti-malignant treatment.
... The AlphaFold model predicts that Hsp90 inhibitors may not bind sacsin because of steric hinderance from Asp 168 Hsp90 can be targeted by drugs that inhibit its ATPase activity through competitive binding of the nucleotide binding pocket (e.g., radicicol, geldanamycin and its analog tanespimycin/17-AAG) (Armstrong et al., 2018). Given the homology between the Hsp90 ATP binding domain and the sacsin SR1 domains there is a possibility these inhibitors, or related drugs, may target sacsin. ...
The ataxia-linked protein sacsin has three regions of partial homology to Hsp90’s N-terminal ATP binding domain. Although a crystal structure for this Hsp90-like domain has been reported the precise molecular interactions required for ATP-binding and hydrolysis are unclear and it is debatable whether ATP biding is compatible with these domains. Furthermore, the Identification of a sacsin domain(s) equivalent to the middle domain of Hsp90 has been elusive. Here we present the superimposition of an AlphaFold structure of sacsin with yeast Hsp90, which provides novel insights into sacsin’s structure. We identify residues within the sacsin Hsp90-like domains that are required for ATP binding and hydrolysis, including the putative catalytic arginine residues equivalent to that of the Hsp90 middle domain. Importantly, our analysis allows comparison of the Hsp90 middle domain with corresponding sacsin regions and identifies a shorter lid segment, in the sacsin ATP-binding domains, than the one found in the N-terminal domain of Hsp90. Our results show how a realignment of residues in the lid segment of sacsin that are involved in ATP binding can better match equivalent residues seen in Hsp90, which we then corroborated using molecular dynamic simulations. We speculate, from a structural viewpoint, why some ATP competitive inhibitors of Hsp90 may not bind sacsin, while others would. Together our analysis supports the hypothesis that sacsin’s function is ATP-driven and would be consistent with it having a role as a super molecular chaperone. We propose that the SR1 regions of sacsin be renamed as HSP-NRD (Hsp90 N-Terminal Repeat Domain; residues 84-324) and the fragment immediately after as HSP-MRD (Hsp90 Middle Repeat Domain; residues 325-518).
... They used 2-DE coupled MS to show that the treatment of SU11274, a cMET inhibitor along with anti-myeloma drugs (Bortezomib and Lenalidomide) downregulates the levels of angiogenic proteins such as annexin A4 (ANXA4) and prohibitin (PHB), peroxiredoxin-6 (PRDX6), and annexin A2 (ANXA2), while it upregulates the level of calpain small subunit 1 (CPNS1). Armstrong et al. used proteomics and transcriptomics combinatorial approaches to investigate the drug action of AUY922 (a next-generation Hsp90 inhibitor) in prostate cancer explants of prostate cancer patients [540,541]. They found that interference of fibronectin, a cytoskeletal and ECM-related protein, by AUY922 decreases the invasive potential of prostate cancer cell lines. ...
Proteomics continues to forge significant strides in the discovery of essential biological processes, uncovering valuable information on the identity, global protein abundance, protein modifications, proteoform levels, and signal transduction pathways. Cancer is a complicated and heterogeneous disease, and the onset and progression involve multiple dysregulated proteoforms and their downstream signaling pathways. These are modulated by various factors such as molecular, genetic, tissue, cellular, ethnic/racial, socioeconomic status, environmental, and demographic differences that vary with time. The knowledge of cancer has improved the treatment and clinical management; however, the survival rates have not increased significantly, and cancer remains a major cause of mortality. Oncoproteomics studies help to develop and validate proteomics technologies for routine application in clinical laboratories for (1) diagnostic and prognostic categorization of cancer, (2) real-time monitoring of treatment, (3) assessing drug efficacy and toxicity, (4) therapeutic modulations based on the changes with prognosis and drug resistance, and (5) personalized medication. Investigation of tumor-specific proteomic profiles in conjunction with healthy controls provides crucial information in mechanistic studies on tumorigenesis, metastasis, and drug resistance. This review provides an overview of proteomics technologies that assist the discovery of novel drug targets, biomarkers for early detection, surveillance, prognosis, drug monitoring, and tailoring therapy to the cancer patient. The information gained from such technologies has drastically improved cancer research. We further provide exemplars from recent oncoproteomics applications in the discovery of biomarkers in various cancers, drug discovery, and clinical treatment. Overall, the future of oncoproteomics holds enormous potential for translating technologies from the bench to the bedside.
... In addition, drugs targeting molecular chaperone-related genes are also widely used in clinical practice. chaperones-related related genes with great clinical potential, such as HSP90 and P53, making immunotherapy become an important means of tumor treatment (Armstrong et al., 2018;Kaida and Iwakuma, 2021). Although molecular chaperones can be used as prognostic indicators in patients with LUAD, the utility of using only a single biomarker is limited. ...
Introduction: Molecular chaperones and long non-coding RNAs (lncRNAs) have been confirmed to be closely related to the occurrence and development of tumors, especially lung cancer. Our study aimed to construct a kind of molecular chaperone-related long non-coding RNAs (MCRLncs) marker to accurately predict the prognosis of lung adenocarcinoma (LUAD) patients and find new immunotherapy targets.
Methods: In this study, we acquired molecular chaperone genes from two databases, Genecards and molecular signatures database (MsigDB). And then, we downloaded transcriptome data, clinical data, and mutation information of LUAD patients through the Cancer Genome Atlas (TCGA). MCRLncs were determined by Spearman correlation analysis. We used univariate, least absolute shrinkage and selection operator (LASSO) and multivariate Cox regression analysis to construct risk models. Kaplan-meier (KM) analysis was used to understand the difference in survival between high and low-risk groups. Nomogram, calibration curve, concordance index (C-index) curve, and receiver operating characteristic (ROC) curve were used to evaluate the accuracy of the risk model prediction. In addition, we used gene ontology (GO) enrichment analysis and kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses to explore the potential biological functions of MCRLncs. Immune microenvironmental landscapes were constructed by using single-sample gene set enrichment analysis (ssGSEA), tumor immune dysfunction and exclusion (TIDE) algorithm, “pRRophetic” R package, and “IMvigor210” dataset. The stem cell index based on mRNAsi expression was used to further evaluate the patient’s prognosis.
Results: Sixteen MCRLncs were identified as independent prognostic indicators in patients with LUAD. Patients in the high-risk group had significantly worse overall survival (OS). ROC curve suggested that the prognostic features of MCRLncs had a good predictive ability for OS. Immune system activation was more pronounced in the high-risk group. Prognostic features of the high-risk group were strongly associated with exclusion and cancer-associated fibroblasts (CAF). According to this prognostic model, a total of 15 potential chemotherapeutic agents were screened for the treatment of LUAD. Immunotherapy analysis showed that the selected chemotherapeutic drugs had potential application value. Stem cell index mRNAsi correlates with prognosis in patients with LUAD.
Conclusion: Our study established a kind of novel MCRLncs marker that can effectively predict OS in LUAD patients and provided a new model for the application of immunotherapy in clinical practice.
... A core biopsy of tissue was dissected into 1-mm 3 pieces and cultured in duplicate using a modified method as previously described. 50,51 Although not as physiologically ideal as directly treating patients with dietary factors, the ex vivo model provides advantages by measuring response of live human biopsies (containing all of the cell types within human intestinal tissues) to treatments in a more controlled environment (e.g., devoid of wide variability in patient diets that may interfere with results analysis). Tissues were cultured on a presoaked gelatin sponge (Surgifoam) in 48-well culture plates containing 250 µL solution (PBS + indicated fiber, or PBS control; 5% DMEM/F12 [with 5% FBS], 100 U/mL penicillin/100 µg/mL streptomycin antibiotic/antimycotic solution). ...
Background and aims: Inflammatory bowel diseases (IBD) are impacted by dietary factors, including non-digestible carbohydrates (fibers), which are fermented by colonic microbes. Fibers are overall beneficial but not all fibers are alike and some IBD patients report intolerance to fiber consumption. Given reproducible evidence of reduced fiber-fermenting microbes in IBD patients, we hypothesized that fibers remain intact in select patients with reduced fiber-fermenting microbes and can then bind host cell receptors, subsequently promoting gut inflammation.
Methods: Colonic biopsies cultured ex vivo and cell lines in vitro were incubated with oligofructose (5g/L), or fermentation supernatants (24hr anaerobic fermentation) and immune responses (cytokine secretion [ELISA/MSD] and expression [qPCR]) were assessed. Influence of microbiota in mediating host response was examined and taxonomic classification of microbiota was conducted with Kraken2 and metabolic profiling by HUMAnN2, using R software.
Results: Unfermented dietary β-fructan fibers induced pro-inflammatory cytokines in a subset of IBD intestinal biopsies cultured ex vivo, and immune cells (including peripheral blood mononuclear cells). Results were validated in an adult IBD randomized controlled trial examining β-fructan supplementation. The pro-inflammatory response to intact β-fructan required activation of the NLRP3 and TLR2 pathways. Fermentation of β-fructans by human gut whole-microbiota cultures reduced the pro-inflammatory response, but only when microbes were collected from non-IBD or inactive IBD patients. Fiber-induced immune responses correlated with microbe functions, luminal metabolites, and dietary fiber avoidance.
Conclusion: While fibers are typically beneficial in individuals with normal microbial fermentative potential, some dietary fibers have detrimental effects in select patients with active IBD who lack fermentative microbe activities.
... Differentially expressed (DE) transcripts in Hsp90 inhibitor treated PDEs were identified by RNA sequencing, as described in our previous report [33]. Proteins in Hsp90 inhibitor treated PDEs were detected using liquid chromatography tandem mass spectrometry (LC-MS/MS) as previously reported [34], and subjected to statistical analysis to detect proteins that were differentially abundant (DA) using a voom-eBayes approach under the paired design (R 3.5.0, ...
... The natural variability between PDE samples means that designing omics experiments with adequate power to detect differential expressions at the level required to answer research questions must be carefully considered. A pilot RNAseq experiment was previously conducted and validated [33], and we now reanalyze this dataset to establish the influence of PDE heterogeneity on transcriptomic analysis. PDEs treated with two molecular therapies targeting Hsp90 were used for the pilot RNAseq study, as we have published the proliferative (Ki67) response of prostate PDEs to highly effective Hsp90 inhibitor AUY922 and the less potent inhibitor 17-AAG [22]. ...
Simple Summary
There is a widespread push toward more biologically relevant pre-clinical models of prostate cancer that can improve the discovery and translation of new drugs and biomarkers for this disease. Patient-derived explant culture is an innovative pre-clinical model that utilizes surgical prostate cancer specimens in a way that retains the architecture, microenvironment and heterogeneity of prostate tumors—factors that critically influence cell behavior and response to therapy. With increasing tissue complexity comes increasing complexity of analysis. The aim of this study was to provide critical information for the successful application and analysis of the patient-derived prostate cancer explant model.
Abstract
Prostate cancer is a complex and heterogeneous disease, but a small number of cell lines have dominated basic prostate cancer research, representing a major obstacle in the field of drug and biomarker discovery. A growing lack of confidence in cell lines has seen a shift toward more sophisticated pre-clinical cancer models that incorporate patient-derived tumors as xenografts or explants, to more accurately reflect clinical disease. Not only do these models retain critical features of the original tumor, and account for the molecular diversity and cellular heterogeneity of prostate cancer, but they provide a unique opportunity to conduct research in matched tumor samples. The challenge that accompanies these complex tissue models is increased complexity of analysis. With over 10 years of experience working with patient-derived explants (PDEs) of prostate cancer, this study provides guidance on the PDE method, its limitations, and considerations for addressing the heterogeneity of prostate cancer PDEs that are based on statistical modeling. Using inhibitors of the molecular chaperone heat shock protein 90 (Hsp90) as an example of a drug that induces robust proliferative response, we demonstrate how multi-omics analysis in prostate cancer PDEs is both feasible and essential for identification of key biological pathways, with significant potential for novel drug target and biomarker discovery.
... HSP90 inhibitors also possess the ability to disrupt vesicular secretory trafficking of the extracellular matrix (ECM), thus reducing fibronectin deposition and tissue remodeling [32]. As shown by in vitro co-immunoprecipitation assay, HSP90 directly binds fibronectin and pharmacological or genetic HSP90 inhibition blocks fibronectin release from cells [33]. ...
... HSP90 inhibitors also possess the ability to disrupt vesicular secretory trafficking of the extracellular matrix (ECM), thus reducing fibronectin deposition and tissue remodeling [32]. ...
Hydrochloric acid (HCl) exposure causes asthma-like conditions, reactive airways dysfunction syndrome, and pulmonary fibrosis. Heat Shock Protein 90 (HSP90) is a molecular chaperone that regulates multiple cellular processes. HSP90 inhibitors are undergoing clinical trials for cancer and are also being studied in various pre-clinical settings for their anti-inflammatory and anti-fibrotic effects. Here we investigated the ability of the heat shock protein 90 (HSP90) inhibitor AT13387 to prevent chronic lung injury induced by exposure to HCl in vivo and its protective role in the endothelial barrier in vitro. We instilled C57Bl/6J mice with 0.1N HCl (2 µL/g body weight, intratracheally) and after 24 h began treatment with vehicle or AT13387 (10 or 15 mg/kg, SC), administered 3×/week; we analyzed histological, functional, and molecular markers 30 days after HCl. In addition, we monitored transendothelial electrical resistance (TER) and protein expression in a monolayer of human lung microvascular endothelial cells (HLMVEC) exposed to HCl (0.02 N) and treated with vehicle or AT13387 (2 µM). HCl provoked persistent alveolar inflammation; activation of profibrotic pathways (MAPK/ERK, HSP90); increased deposition of collagen, fibronectin and elastin; histological evidence of fibrosis; and a decline in lung function reflected in a downward shift in pressure–volume curves, increased respiratory system resistance (Rrs), elastance (Ers), tissue damping (G), and hyperresponsiveness to methacholine. Treatment with 15 mg/kg AT13387reduced alveolar inflammation, fibrosis, and NLRP3 staining; blocked activation of ERK and HSP90; and attenuated the deposition of collagen and the development of chronic lung injury and airway hyperreactivity. In vitro, AT13387 prevented HCl-induced loss of barrier function and AKT, ERK, and ROCK1 activation, and restored HSP70 and cofilin expression. The HSP90 inhibitor, AT13387, represents a promising drug candidate for chronic lung injury that can be administered subcutaneously in the field, and at low, non-toxic doses.
