BACKGROUND: Cancer stem-like cells (CSCs) represent a subset of tumor cells that have the ability to self-renew, a long lifespan and a relatively quiescent phenotype, and show resistance to conventional therapies. Various markers are used to identify CSCs, and have shown that different CSC subtypes may be present within a tumor. One functional property of CSCs is their relative lack of proteasomal activity compared to the tumor bulk. METHODS: We introduced an unstable fluorescent molecule into FaDu oropharyngeal squamous cell carcinoma cells and analyzed the association of proteasome activity with aldehydehyde dehydrogenase (ALDH) activity as another common CSC marker, and with other stem-cell related properties of glucose metabolism. We also analyzed publicly available gene expression profiling data of ALDH+ CSCs for alterations in mRNAs associated with proteostasis. RESULTS: We show that FaDu CSCs identified by low proteasome activity are associated with the population identified by high ALDH activity. Futher characterization shows that these CSCs have a relatively high mitochondrial membrane potential and low levels of glucose transporter, indicating a non-Warburg metabolic phenotype. We also show that proteasome-low FaDu CSCs exhibit decreased rates of protein synthesis. Gene expression profiling of other cancer cell lines reveal common statistically significant differences in proteostasis in ALDH+ CSCs compared to the bulk of the tumor cells, including reduced levels of Hsp70 and/or Hsp90 in CSCs defined by ALDH, together with reduced levels of UCHL5 mRNA. CONCLUSIONS: These data provide additional insights into the functional characteristics of proteasome-low/ALDH-high CSCs, indicating a metabolic phenotype of reduced reliance on aerobic glycolysis and a decreased protein synthesis rate. We also identify specific chaperone and ubiquitin ligase activities that can be used to identify CSCs, with corresponding implications for therapeutic strategies that target CSCs through their altered metabolic properties.

• Keywords
• Cancer stem cells, Glucose transporter, Mitochondrial membrane potential, Protein degradation, Proteosynthesis, Squamous cell carcinoma,
• MeSH
• Aldehyde Dehydrogenase * metabolism   genetics   MeSH
• Glucose metabolism   MeSH
• Humans MeSH
• Membrane Potential, Mitochondrial MeSH
• Mitochondria * metabolism   MeSH
• Cell Line, Tumor MeSH
• Neoplastic Stem Cells * metabolism   pathology   MeSH
• Proteasome Endopeptidase Complex metabolism   MeSH
• Glucose Transport Proteins, Facilitative * metabolism   MeSH
• Gene Expression Regulation, Neoplastic MeSH
• Carcinoma, Squamous Cell * metabolism   pathology   genetics   MeSH
• Gene Expression Profiling MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Aldehyde Dehydrogenase * MeSH
• Glucose MeSH
• Proteasome Endopeptidase Complex MeSH
• Glucose Transport Proteins, Facilitative * MeSH

Background: A hallmark of cancer is the presence of an immunosuppressive tumor microenvironment (TME). Immunosuppressive M2 macrophages (MΦs) in the TME facilitate escape from immune surveillance and promote tumor growth; therefore, TME-induced immunosuppression is a potent immunotherapeutic approach to treating cancer. Methods: Cancer cell-secreted proteins were detected by using liquid chromatography-mass spectrometry (LC-MS). Neutralizing antibodies (nAbs) were used to assess which proteins were involved in MΦs polarization and differentiation. The protein-protein interaction was characterized using co-immunoprecipitation and immunofluorescence assays. Cancer-secreted heat shock protein 70 (Hsp70) protein was quantified using an enzyme-linked immunosorbent assay (ELISA). MΦ polarization and tumor growth were assessed in vivo with subcutaneous LLC-GFP tumor models and toll-like receptor 2 (TLR2) knockout mice; in vitro assessments were conducted using TLR2 knockout and both LLC-GFP and LN227 lentiviral-mediated knockdown (KD) cells. Results: Cancer cells released a secreted form of Hsp70 that acted on MΦ TLR2 to upregulate Mer receptor tyrosine kinase (MerTK) and induce MΦ M2 polarization. Hsp70 nAbs led to a reduction in CD14 expression by 75% in THP-1 cells in response to Gli36 EMD-CM. In addition, neutralizing TLR2 nAbs resulted in a 30% and 50% reduction in CD14 expression on THP-1 cells in response to MiaPaCa-2 and Gli36 exosome/microparticle-depleted conditioned media (EMD-CMs), respectively. Hsp70, TLR2, and MerTK formed a protein complex. Tumor growth and intra-tumor M2 MΦs were significantly reduced upon cancer cell Hsp70 knockdown and in TLR2 knockout mice. Conclusions: Cancer-secreted Hsp70 interacts with TLR2, upregulates MerTK on MΦs, and induces immunosuppressive MΦ M2 polarization. This previously unreported action of secreted Hsp70 suggests that disrupting the Hsp70-TLR2-MerTK interaction could serve as a promising immunotherapeutic approach to mitigate TME immunosuppression in solid cancers.

• Keywords
• Hsp70, M2 macrophage polarization, MerTK, TLR2, cancer, phospho-Hsp70,
• Publication type
• Journal Article MeSH

Translocase of outer mitochondrial membrane 34 (TOMM34) orchestrates heat shock protein 70 (HSP70)/HSP90-mediated transport of mitochondrial precursor proteins. Here, using in vitro phosphorylation and refolding assays, analytical size-exclusion chromatography, and hydrogen/deuterium exchange MS, we found that TOMM34 associates with 14-3-3 proteins after its phosphorylation by protein kinase A (PKA). PKA preferentially targeted two serine residues in TOMM34: Ser93 and Ser160, located in the tetratricopeptide repeat 1 (TPR1) domain and the interdomain linker, respectively. Both of these residues were necessary for efficient 14-3-3 protein binding. We determined that phosphorylation-induced structural changes in TOMM34 are further augmented by binding to 14-3-3, leading to destabilization of TOMM34's secondary structure. We also observed that this interaction with 14-3-3 occludes the TOMM34 interaction interface with ATP-bound HSP70 dimers, which leaves them intact and thereby eliminates an inhibitory effect of TOMM34 on HSP70-mediated refolding in vitro In contrast, we noted that TOMM34 in complex with 14-3-3 could bind HSP90. Both TOMM34 and 14-3-3 participated in cytosolic precursor protein transport mediated by the coordinated activities of HSP70 and HSP90. Our results provide important insights into how PKA-mediated phosphorylation and 14-3-3 binding regulate the availability of TOMM34 for its interaction with HSP70.

• Keywords
• 14-3-3 protein, 70-kDa heat shock protein (Hsp70), HSP70, Hsp70, Tomm34, dimerization, hydrogen-deuterium exchange, molecular chaperone, phosphorylation, protein folding, protein import, protein kinase A (PKA), protein-nucleic acid interaction, protein–protein interaction, translocase of outer mitochondrial membrane 34 (TOMM34),
• MeSH
• DNA-Binding Proteins genetics   metabolism   MeSH
• Phosphorylation physiology   MeSH
• Humans MeSH
• MCF-7 Cells MeSH
• Mitochondrial Precursor Protein Import Complex Proteins MeSH
• Mitochondrial Membranes metabolism   MeSH
• Mitochondrial Proteins metabolism   MeSH
• Molecular Chaperones metabolism   MeSH
• Cyclic AMP-Dependent Protein Kinases metabolism   MeSH
• 14-3-3 Proteins metabolism   MeSH
• HSP70 Heat-Shock Proteins metabolism   MeSH
• HSP72 Heat-Shock Proteins metabolism   MeSH
• HSP90 Heat-Shock Proteins metabolism   MeSH
• Signal Transduction MeSH
• Transcription Factors genetics   metabolism   MeSH
• Mitochondrial Membrane Transport Proteins genetics   metabolism   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• BCL2-associated athanogene 1 protein MeSH Browser
• DNA-Binding Proteins MeSH
• Mitochondrial Precursor Protein Import Complex Proteins MeSH
• Mitochondrial Proteins MeSH
• Molecular Chaperones MeSH
• Cyclic AMP-Dependent Protein Kinases MeSH
• 14-3-3 Proteins MeSH
• HSP70 Heat-Shock Proteins MeSH
• HSP72 Heat-Shock Proteins MeSH
• HSP90 Heat-Shock Proteins MeSH
• TOMM34 protein, human MeSH Browser
• Transcription Factors MeSH
• Mitochondrial Membrane Transport Proteins MeSH

BACKGROUND: Increased activity of the chaperones Hsp70 and Hsp90 is a common feature of solid tumours. Translocase of the outer mitochondrial membrane 34 (Tomm34) is a cochaperone of both Hsp70 and Hsp90 that was found to be overexpressed in colorectal, hepatocellular, lung and breast carcinomas. The expression profile of Tomm34 in ovarian cancer has not been investigated. Therefore, the aim of the current study was to investigate the expression pattern of Tomm34 in ovarian carcinomas and analyse its correlation with clinico-pathological parameters. RESULTS: Epithelial ovarian cancers (140) were histologically classified based on their morphology and graded into two types comprising 5 histologic subgroups. Type I carcinomas comprise low grade serous (LGSC), clear cell (CCOC) and endometrioid (ENOC), type II comprises high grade serous carcinomas (HGSC) and solid, pseudoendometrioid, transitional carcinomas (SET). Tomm34 was more highly expressed in type II than type I carcinomas (p < 0.0001). Comparing tumours based on the mutation in the TP53 gene revealed similar results, where mutant tumours exhibited significantly higher levels of Tomm34 (p < 0.0001). The decreased levels of Tomm34 in type I carcinomas were particularly evident in clear cell and mucinous carcinomas. The expression of Tomm34 was also positively correlated with FIGO stage (r = 0.23; p = 0.007). Tomm34 levels also indicated poor prognosis for patients with mutant p53. CONCLUSIONS: Our data indicate that Tomm34 is commonly expressed at high levels in epithelial ovarian cancers, except for the clear cell and mucinous subtypes. The expression of Tomm34 corresponds with the dualistic model of ovarian cancer pathogenesis where high grade, type II tumours exhibit higher expression of Tomm34 in contrast to type I tumours. These data are also comparable to the previous findings that Tomm34 is a marker of progression and poor prognosis in human cancer.

• Keywords
• Chaperone, Epithelial ovarian cancer, Heat shock protein, Immunohistochemistry, Ovary, Tomm34, Tumour,
• MeSH
• Adult MeSH
• Carcinoma, Ovarian Epithelial metabolism   pathology   MeSH
• Immunohistochemistry MeSH
• Middle Aged MeSH
• Humans MeSH
• Mitochondrial Precursor Protein Import Complex Proteins MeSH
• Mutation MeSH
• Biomarkers, Tumor genetics   metabolism   MeSH
• Tumor Suppressor Protein p53 genetics   MeSH
• Ovarian Neoplasms metabolism   pathology   MeSH
• Prognosis MeSH
• Disease Progression MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Neoplasm Staging MeSH
• Mitochondrial Membrane Transport Proteins metabolism   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Mitochondrial Precursor Protein Import Complex Proteins MeSH
• Biomarkers, Tumor MeSH
• Tumor Suppressor Protein p53 MeSH
• TOMM34 protein, human MeSH Browser
• TP53 protein, human MeSH Browser
• Mitochondrial Membrane Transport Proteins MeSH

Response of tumours to Hsp90 inhibitors is highly variable and their clinical effects are unpredictable, emphasising the need for a predictive marker. We postulated that sensitivity to Hsp90 inhibitors is connected to basal proteotoxic stress that makes cells dependent on Hsp90. Therefore, we assessed HSF1 as a general sensor of proteotoxic stress and correlated its activity with sensitivity to three separate small molecule Hsp90 inhibitors in seven breast cancer cell lines representing each of the different cancer subtypes. Flow cytometry was used to analyse the viability of breast cancer cell lines after Hsp90 inhibition. HSF1 activity was characterised by Ser326 phosphorylation and the transactivation capacity of HSF1 was determined by qPCR analysis of the ratios of HSF1-dependent (HOP, Hsp70) and HSF1-independent (CHIP) chaperones and cochaperone mRNAs. We show that the sensitivity of breast cancer cell lines to Hsp90 inhibition is highly variable. The basal levels of phosphorylated HSF1 also vary between cell lines and the magnitude of change in HSF1 phosphorylation after Hsp90 inhibition showed a negative correlation with sensitivity to Hsp90 inhibitors. Similarly, the basal transactivation capacity of HSF1, determined by the ratio of Hsp70 or HOP mRNA to CHIP mRNA level, is directly proportional to sensitivity to Hsp90 inhibitors. Increasing basal HSF1 activity by prior heat shock sensitised cells to Hsp90 inhibition. These results demonstrate that endogenous HSF1 activity varies between individual cancer cell lines and inversely reflects their sensitivity to Hsp90 inhibitors, suggesting that basal proteotoxic stress is an important and generalised predictor of response. Mechanistically, the data indicate that high endogenous proteotoxic stress levels sensitise to Hsp90 inhibition due to the inability to respond adequately to further proteotoxic stress. HSF1 activity therefore represents a potential biomarker for therapy with Hsp90 inhibitors, which may be useful for the rational design of future clinical studies.

• MeSH
• Biomarkers metabolism   MeSH
• Phosphorylation drug effects   MeSH
• Small Molecule Libraries pharmacology   MeSH
• Humans MeSH
• MCF-7 Cells MeSH
• Molecular Chaperones genetics   MeSH
• Cell Line, Tumor MeSH
• Breast Neoplasms drug therapy   metabolism   MeSH
• Cell Proliferation drug effects   MeSH
• HSP90 Heat-Shock Proteins antagonists & inhibitors   MeSH
• Heat-Shock Response MeSH
• Heat Shock Transcription Factors metabolism   MeSH
• Cell Survival drug effects   MeSH
• Check Tag
• Humans MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Biomarkers MeSH
• HSF1 protein, human MeSH Browser
• Small Molecule Libraries MeSH
• Molecular Chaperones MeSH
• HSP90 Heat-Shock Proteins MeSH
• Heat Shock Transcription Factors MeSH

CHIP is a tetratricopeptide repeat (TPR) domain protein that functions as an E3-ubiquitin ligase. As well as linking the molecular chaperones to the ubiquitin proteasome system, CHIP also has a docking-dependent mode where it ubiquitinates native substrates, thereby regulating their steady state levels and/or function. Here we explore the effect of Hsp70 on the docking-dependent E3-ligase activity of CHIP. The TPR-domain is revealed as a binding site for allosteric modulators involved in determining CHIP's dynamic conformation and activity. Biochemical, biophysical and modeling evidence demonstrate that Hsp70-binding to the TPR, or Hsp70-mimetic mutations, regulate CHIP-mediated ubiquitination of p53 and IRF-1 through effects on U-box activity and substrate binding. HDX-MS was used to establish that conformational-inhibition-signals extended from the TPR-domain to the U-box. This underscores inter-domain allosteric regulation of CHIP by the core molecular chaperones. Defining the chaperone-associated TPR-domain of CHIP as a manager of inter-domain communication highlights the potential for scaffolding modules to regulate, as well as assemble, complexes that are fundamental to protein homeostatic control.

• MeSH
• Allosteric Regulation MeSH
• Gene Expression MeSH
• Interferon Regulatory Factor-1 genetics   metabolism   MeSH
• Kinetics MeSH
• Humans MeSH
• Lymphocytes cytology   metabolism   MeSH
• Protein Interaction Mapping MeSH
• Models, Molecular MeSH
• Cell Line, Tumor MeSH
• Tumor Suppressor Protein p53 genetics   metabolism   MeSH
• Proteasome Endopeptidase Complex metabolism   MeSH
• HSP70 Heat-Shock Proteins chemistry   genetics   metabolism   MeSH
• Protein Structure, Secondary MeSH
• Protein Structure, Tertiary MeSH
• Ubiquitination MeSH
• Ubiquitin-Protein Ligases chemistry   genetics   metabolism   MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Interferon Regulatory Factor-1 MeSH
• Tumor Suppressor Protein p53 MeSH
• Proteasome Endopeptidase Complex MeSH
• HSP70 Heat-Shock Proteins MeSH
• STUB1 protein, human MeSH Browser
• Ubiquitin-Protein Ligases MeSH