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This project aims to investigate the release performance of bilayer tablet (BL-Tablet) designed with both fast and slow-release technology, targeting sleep disorders. The tablet incorporates Melatonin, extracts of Eschscholzia californica and Melissa officinalis . In order to validate the effectiveness of the extended-release profile, an advanced dissolution test was herein proposed. This new method utilizes biorelevant intestinal fluid media and incorporates a stomach-to-intestine fluid changing (SIFC) system. To demonstrate the advantages of employing this method for assessing the controlled release profile of active ingredients, the dissolution results were compared with those obtained using the conventional EU Pharmacopoeia approach. Furthermore, the comparative analysis was extended to include a monolayer tablet version (ML-Tablet) lacking the slow-release technology. Technological characterization and bioaccessibility studies, including intestinal permeability test, were conducted as well to assess the pharmacological performance and bioavailability of active ingredients. The dissolution data recovered revealed that the two dissolution methods did not exhibit any significant differences in the release of ML-Tablet's. However, the dissolution profile of the BL-Tablet exhibited notable differences between the two methods particularly when assessing the behavior of the slow-release layer. In this scenario, both methods initially exhibited a similar release pattern within the first approximately 0.5 h, driven by the fast-release layer of the tablet. Following this, distinct gradual and sustained releases were observed, spanning 2.5 h for the EU Pharmacopoeia method and 8 h for the new SIFC-biorelevant dissolution method, respectively. Overall, the novel method demonstrated a substantial improvement compared to conventional EU Pharmacopoeia test in evaluating the performance of a controlled slow-release technology. Remarkably, the prolonged release technology did not have an adverse impact on melatonin intestinal absorption, and, consequently, maintaining its potential bioavailability of around 78%. Concluding, this research provides valuable insights into how the innovative dissolution test can assist formulators in developing controlled release formulations., Competing Interests: RB, EA, FC, SP, PI, PG, MC, SF, JG, SZ, and WB were employed by Labomar SpA., (Copyright © 2024 Bassetto, Amadio, Ciampanelli, Perin, Ilari, Gaballo, Callegari, Feltrin, Gobbo, Zanatta and Bertin.)

The 3 d transition metal (Mn(II), Fe(II), Co(II), Ni(II), Cu(II) and Zn(II)) complexes, supported by anions of sterically demanding β-diketones, 1,3-dimesitylpropane-1,3-dione (HL Mes ) and 1,3-bis(3,5-bis(trifluoromethyl)phenyl)-3-hydroxyprop-2-en-1-one (HL CF3 ), were synthesized and evaluated for their antitumor activity. To assess the biological effects of substituents on phenyl moieties, we also synthesized and investigated the analogous metal(II) complexes of the anion of the less bulky 1,3-diphenylpropane-1,3-dione (HL Ph ) ligand. The compounds [Cu(L CF3 ) 2 ], [Cu(L Mes ) 2 ] and ([Zn(L Mes ) 2 ]) were characterized by X-ray crystallography. The [Cu(L CF3 ) 2 ] crystallizes with an apical molecule of solvent (THF) and features a rare square pyramidal geometry at the Cu(II) center. The copper(II) and zinc(II) complexes of diketonate ligands, derived from the deprotonated 1,3-dimesitylpropane-1,3-dione (HL Mes ), adopt a square planar or a tetrahedral geometry at the metal, respectively. We evaluated the antitumor properties of the newly synthesized (Mn(II), Fe(II), Co(II), Ni(II), Cu(II) and Zn(II)) complexes against a series of human tumor cell lines derived from different solid tumors. Except for iron derivatives, cellular studies revealed noteworthy antitumor properties, even towards cancer cells endowed with poor sensitivity to the reference drug cisplatin.

The new 3-monosubstituted acetylacetone ligands, 3-(phenyl(1 H -pyrazol-1-yl)methyl)pentane-2,4-dione (HL acPz ) and 3-((3,5-dimethyl-1 H -pyrazol-1-yl)(phenyl)methyl)pentane-2,4-dione (HL acPzMe ), were synthesized and used as supporting ligands for new copper(II) and copper(I) phosphane complexes of the general formulae [Cu(HL acX ) 2 (L acX ) 2 ] and [Cu(PPh 3 ) 2 (HL acX )]PF 6 (X = Pz (pyrazole) or PzMe (3,5-dimethylpyrazole)), respectively. In the syntheses of the Cu(I) complexes, the triphenylphosphine coligand (PPh 3 ) was used to stabilize copper in the +1 oxidation state, avoiding oxidation to Cu(II). All compounds were characterized by CHN analysis, 1 H-NMR, 13 C-NMR, FT-IR spectroscopy, and electrospray ionization mass spectrometry (ESI-MS). The ligands HL acPz ( 1 ) and HL acPzMe ( 2 ) and the copper complex [Cu(PPh 3 ) 2 (HL acPz )]PF 6 ( 3 ) were also characterized by X-ray crystallography. The reactivity of these new compounds was investigated and the new compounds 4-phenyl-4-(1 H -pyrazol-1-yl)butan-2-one ( 7 ) and 4-(3,5-dimethyl-1 H -pyrazol-1-yl)-4-phenylbutan-2-one ( 8 ) were obtained in basic conditions via the retro-Claisen reaction of related 3-monosubstituted acetylacetone, providing efficient access to synthetically useful ketone compounds. Compound 8 was also characterized by X-ray crystallography.

Background: Circulating tumour cells (CTCs), circulating tumour DNA (ctDNA), and extracellular vesicles (EVs) are minimally invasive liquid biopsy biomarkers. This study investigated whether they predict prognosis, alone or in combination, in heterogenous unbiased non-small cell lung cancer (NSCLC) patients., Methods: Plasma samples of 54 advanced NSCLC patients from a prospective clinical trial. CtDNA mutations were identified using the UltraSEEK™ Lung Panel (MassARRAY® technology). PD-L1 expression was assessed in small EVs (sEVs) using an enzyme-linked immunosorbent assay., Results: At least one ctDNA mutation was detected in 37% of patients. Mutations were not correlated with overall survival (OS) (HR = 1.1, 95% CI = 0.55; 1.83, P = 0.980) and progression-free survival (PFS) (HR = 1.00, 95% CI = 0.57-1.76, P = 0.991). High PD-L1 + sEV concentration was correlated with OS (HR = 1.14, 95% CI = 1.03-1.26, P = 0.016), but not with PFS (HR = 1.08, 95% CI = 0.99-1.18, P = 0.095). The interaction analysis suggested that PD-L1 + sEV correlation with PFS changed in function of CTC presence/absence (P interaction = 0.036). The combination analysis highlighted worse prognosis for patients with CTCs and high PD-L1 + sEV concentration (HR = 7.65, 95% CI = 3.11-18.83, P < 0.001). The mutational statuses of ctDNA and tumour tissue were significantly correlated (P = 0.0001)., Conclusion: CTCs and high PD-L1 + sEV concentration correlated with PFS and OS, but not ctDNA mutations. Their combined analysis may help to identify patients with worse OS., Trial Registration: NCT02866149, Registered 01 June 2015, https://clinicaltrials.gov/ct2/show/study/NCT02866149 ., (© 2023. The Author(s).)

Patients carrying autosomal dominant mutations in the histone/lysine acetyl transferases CBP or EP300 develop a neurodevelopmental disorder: Rubinstein-Taybi syndrome (RSTS). The biological pathways underlying these neurodevelopmental defects remain elusive. Here, we unravel the contribution of a stress-responsive pathway to RSTS. We characterize the structural and functional interaction between CBP/EP300 and heat-shock factor 2 (HSF2), a tuner of brain cortical development and major player in prenatal stress responses in the neocortex: CBP/EP300 acetylates HSF2, leading to the stabilization of the HSF2 protein. Consequently, RSTS patient-derived primary cells show decreased levels of HSF2 and HSF2-dependent alteration in their repertoire of molecular chaperones and stress response. Moreover, we unravel a CBP/EP300-HSF2-N-cadherin cascade that is also active in neurodevelopmental contexts, and show that its deregulation disturbs neuroepithelial integrity in 2D and 3D organoid models of cerebral development, generated from RSTS patient-derived iPSC cells, providing a molecular reading key for this complex pathology., (© 2022. The Author(s).)

Strong evidence suggests that differences in the molecular composition of lipids in exosomes depend on the cell type and has an influence on cancer initiation and progression. Here, we analyzed by liquid chromatography-mass spectrometry (LC-MS) the lipidomic signature of exosomes derived from the human cell lines normal colon mucosa (NCM460D), and colorectal cancer (CRC) nonmetastatic (HCT116) and metastatic (SW620), and exosomes isolated from the plasma of nonmetastatic and metastatic CRC patients and healthy donors. Analysis of this exhaustive lipid study highlighted changes in some molecular species that were found in the cell lines and confirmed in the patients. For example, exosomes from primary cancer patients and nonmetastatic cells compared with healthy donors and control cells displayed a common marked increase in phosphatidylcholine (PC) 34 : 1, phosphatidylethanolamine (PE) 36 : 2, sphingomyelin (SM) d18 : 1/16 : 0, hexosylceramide (HexCer) d18 : 1/24 : 0 and HexCer d18 : 1/24 : 1. Interestingly, these same lipids species were decreased in the metastatic cell line and patients. Further, levels of PE 34 : 2, PE 36 : 2, and phosphorylated PE p16 : 0/20 : 4 were also significantly decreased in metastatic conditions when compared to the nonmetastatic counterparts. The only molecule species found markedly increased in metastatic conditions (in both patients and cells) when compared to controls was ceramide (Cer) d18 : 1/24 : 1. These decreases in lipid species in the extracellular vesicles might reflect function-associated changes in the metastatic cell membrane. Although these potential biomarkers need to be validated in a larger cohort, they provide new insight toward the use of clusters of lipid biomarkers rather than a single molecule for the diagnosis of different stages of CRC., (© 2022 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Background: Nowadays, evaluation of the efficacy and the duration of treatment, in context of monitoring patients with solid tumors, is based on the RECIST methodology. With these criteria, resistance and/or insensitivity are defined as tumor non-response which does not allow a good understanding of the diversity of the underlying mechanisms. The main objective of the OncoSNIPE® collaborative clinical research program is to identify early and late markers of resistance to treatment., Methods: Multicentric, interventional study with the primary objective to identify early and / or late markers of resistance to treatment, in 600 adult patients with locally advanced or metastatic triple negative or Luminal B breast cancer, non-small-cell lung cancer or pancreatic ductal adenocarcinoma. Patients targeted in this study have all rapid progression of their pathology, making it possible to obtain models for evaluating markers of early and / or late responses over the 2-year period of follow-up, and thus provide the information necessary to understand resistance mechanisms. To explore the phenomena of resistance, during therapeutic response and / or progression of the pathology, we will use a multidisciplinary approach including high-throughput sequencing (Exome-seq and RNAseq), clinical data, medical images and immunological profile by ELISA. Patients will have long-term follow-up with different biological samples, at baseline (blood and biopsy) and at each tumoral evaluation or tumoral progression evaluated by medical imaging. Clinical data will be collected through a dedicated Case Report Form (CRF) and enriched by semantic extraction based on the French ConSoRe (Continuum Soins Recherche) initiative, a dedicated Semantic Clinical Data Warehouse (SCDW) to cancer. The study is sponsored by Oncodesign (Dijon, France) and is currently ongoing., Discussion: The great diversity of intrinsic or acquired molecular mechanisms involved in resistance to treatment constitutes a real therapeutic issue. Improving understanding of mechanisms of resistance of cancer cells to anti-tumor treatments is therefore a major challenge. The OncoSNIPE cohort will lead to a better understanding of the mechanisms of resistance and will allow to explore new mechanisms of actions and to discover new therapeutic targets or strategies making it possible to circumvent the escape in different types of cancer., Trial Registration: Clinicaltrial.gov. Registered 16 September 2020, https://clinicaltrials.gov/ct2/show/NCT04548960?term=oncosnipe&draw=2&rank=1 and ANSM ID RCB 2017-A02018-45., (© 2022. The Author(s).)

Recently, immunotherapy has garnered increasing importance in cancer therapy, leading to substantial improvements in patient care and survival. By blocking the immune checkpoints-protein regulators of the immune system-immunotherapy prevents immune tolerance toward tumors and reactivates the immune system, prompting it to fight cancer cell growth and diffusion. A widespread strategy for this is the blockade of the interaction between PD-L1 and PD-1. However, while patients generally respond well to immunotherapy, a certain proportion of patients present tumors that resist these treatments. This portion can be very high in some cancers and hinders cancer curability. For this reason, current efforts are focusing on combining PD-1/PD-L1 immunotherapy with the targeting of other immune checkpoints to counter resistance and achieve better results. Exosomes, small vesicles secreted by almost any cell, including tumor cells, have proven to be key actors in this resistance. The exosomes released by tumor cells spread the immune-suppressive properties of the tumor throughout the tumor microenvironment and participate in establishing metastatic niches. In this review, we will describe immune checkpoints and immune modulators whose presence in tumor-derived exosomes (TEXs) has been established. We will focus on the most promising proteins under scrutiny for use in combination with PD-1 blockade therapy in a clinical setting, such as PD-L1, CTLA-4, TIM-3, CD73/39, LAG-3, and TIGIT. We will explore the immunosuppressive impact of these exosomal proteins on a variety of immune cells. Finally, we will discuss how they can change the game in immunotherapy and guide therapeutic decisions, as well as the current limits of this approach. Depending on the viewpoint, these exosomal proteins may either provide key missing information on tumor growth and resistance mechanisms or they may be the next big challenge to overcome in improving cancer treatment.

Exosomes are nanovesicles released by all cells that can be found in the blood. A key point for their use as potential biomarkers in cancer is to differentiate tumour-derived exosomes from other circulating nanovesicles. Heat shock protein-70 (HSP70) has been shown to be abundantly expressed by cancer cells and to be associated with bad prognosis. We previously showed that exosomes derived from cancer cells carried HSP70 in the membrane while those from non-cancerous cells did not. In this work, we opened a prospective clinical pilot study including breast and lung cancer patients to determine whether it was possible to detect and quantify HSP70 exosomes in the blood of patients with solid cancers. We found that circulating exosomal HSP70 levels, but not soluble HSP70, reflected HSP70 content within the tumour biopsies. Circulating HSP70 exosomes increased in metastatic patients compared to non-metastatic patients or healthy volunteers. Further, we demonstrated that HSP70-exosome levels correlated with the disease status and, when compared with circulating tumour cells, were more sensitive tumour dissemination predictors. Finally, our case studies indicated that HSP70-exosome levels inversely correlated with response to the therapy and that, therefore, monitoring changes in circulating exosomal HSP70 might be useful to predict tumour response and clinical outcome., (© 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of The International Society for Extracellular Vesicles.)

Background: Cancer is the second leading cause of death globally. Early detection and disease management lead to a better survival rate. Consequently, discovery of novel methods in cancer early diagnosis is a field of active research. Minimally invasive liquid biopsies are generating growing interest. Circulating tumour cells (CTCs) have been identified in patients' blood; nevertheless, these cells are rare and heterogeneous. Exosomes are extracellular nanovesicles released into the extracellular environment via the endosomal vesicle pathway and found in different body fluids. Exosomes deliver bioactive cargo such as proteins, mRNA and miRNA to recipient cells in the tumour environment. We have recently shown that heat shock protein 70 (HSP70) is detected in the membrane of tumour-derived exosomes, in contrast to normal cells. One single cancer cell can release thousands of HSP70-exosomes, facilitating detection. The aim of the pilot study ExoDiag is to determine whether it is possible to detect and quantify HSP70-exosomes in blood in patients with solid cancers., Methods: Bicentric pilot study that will include 60 adult patients with metastatic and non-metastatic solid tumours and 20 healthy volunteers. Exosomes will be isolated from blood and urine samples, and HSP70 concentration will be determined. Patients will be followed for 1 year. The study is sponsored by Georges-François Leclerc Centre and is currently ongoing., Discussion: We expect to demonstrate that HSP70-exosomes could be a powerful tool to diagnose cancer and to guide clinicians in therapeutic decision-making, improving patient's care., Trial Registration: ClinicalTrials.gov identifier NCT02662621. Registered 20 January 2016, https://clinicaltrials.gov/ct2/show/study/NCT02662621?term=NCT02662621&rank=1., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2020.)

In the era of immunotherapies there is an urgent need to implement the use of circulating biomarkers in clinical practice to facilitate personalized therapy and to predict treatment response. We conducted a prospective study to evaluate the usefulness of circulating exosomal-PD-L1 in melanoma patients' follow-up. We studied the dynamics of exosomal-PD-L1 from 100 melanoma patients by using an enzyme-linked immunosorbent assay. We found that PD-L1 was secreted through exosomes by melanoma cells. Exosomes carrying PD-L1 had immunosuppressive properties since they were as efficient as the cancer cell from which they derive at inhibiting T-cell activation. In plasma from melanoma patients, the level of PD-L1 (n= 30, median 64.26 pg/mL) was significantly higher in exosomes compared to soluble PD-L1 (n= 30, 0.1 pg/mL). Furthermore, exosomal-PD-L1 was detected in all patients whereas only 67% of tumour biopsies were PD-L1 positive. Although baseline exosomal-PD-L1 levels were not associated with clinic-pathologic characteristics, their variations after the cures (ΔExoPD-L1) correlated with the tumour response to treatment. A ΔExoPD-L1 cut-off of> 100 was defined, yielding an 83% sensitivity, a 70% specificity, a 91% positive predictive value and 54% negative predictive values for disease progression. The use of the cut-off allowed stratification in two groups of patients statistically different concerning overall survival and progression-free survival. PD-L1 levels in circulating exosomes seem to be a more reliable marker than PD-L1 expression in tumour biopsies. Monitoring of circulating exosomal-PD-L1 may be useful to predict the tumour response to treatment and clinical outcome., (© 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Colorectal cancer (CRC) is one of the major causes of cancer-related deaths worldwide. Tumor microenvironment (TME) contains many cell types including stromal cells, immune cells, and endothelial cells. The TME modulation explains the heterogeneity of response to therapy observed in patients. In this context, exosomes are emerging as major contributors in cancer biology. Indeed, exosomes are implicated in tumor proliferation, angiogenesis, invasion, and premetastatic niche formation. They contain bioactive molecules such as proteins, lipids, and RNAs. More recently, many studies on exosomes have focused on miRNAs, small noncoding RNA molecules able to influence protein expression. In this review, we describe miRNAs transported by exosomes in the context of CRC and discuss their influence on TME and their potential as circulating biomarkers. This overview underlines emerging roles for exosomal miRNAs in cancer research for the near future., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2019 Valentin Vautrot et al.)

A multicenter clinical study demonstrated the presence of a loss-of-function HSP110 mutation in about 15% of colorectal cancers, which resulted from an alternative splicing and was produced at the detriment of wild-type HSP110. Patients expressing low levels of wild-type HSP110 had excellent outcomes (i.e. response to an oxaliplatin-based chemotherapy). Here, we show in vitro, in vivo, and in patients' biopsies that HSP110 co-localizes with DNA damage (γ-H2AX). In colorectal cancer cells, HSP110 translocates into the nucleus upon treatment with genotoxic chemotherapy such as oxaliplatin. Furthermore, we show that HSP110 interacts with the Ku70/Ku80 heterodimer, an essential element of the non-homologous end joining (NHEJ) repair machinery. We also demonstrate by evaluating the resolved 53BP1 foci that depletion in HSP110 impairs repair steps of the NHEJ pathway, which is associated with an increase in DNA double-strand breaks and in the cells' sensitivity to oxaliplatin. HSP110-depleted cells sensitization to oxaliplatin-induced DNA damage is abolished upon re-expression of HSP110. Confirming a role for HSP110 in DNA non-homologous repair, SCR7 and NU7026, two inhibitors of the NHEJ pathway, circumvents HSP110-induced resistance to chemotherapy. In conclusion, HSP110 through its interaction with the Ku70/80 heterodimer may participate in DNA repair, thereby inducing a protection against genotoxic therapy.

Inwardly rectifying potassium channels (Kir), and especially the barium-sensitive Kir4.1 encoded by KCNJ10, are key regulators of glial functions. A lower expression or mislocation of Kir4.1 is detected in human brain tumors. MicroRNAs participate in the regulation of ionic channels and associated neurologic disorders. Here, we analyze effects of miR-5096 on the Kir4.1 expression and function in two glioblastoma cell lines, U87 and U251. Using whole-cell patch-clamp and western-blot analysis, we show that cell loading with miR-5096 decreases the Kir4.1 protein level and associated K+ current. Cell treatment with barium, a Kir4.1 blocker, or cell loading of miR-5096 both increase the outgrowth of filopodia in glioma cells, as observed by time-lapse microscopy. Knocking-down Kir4.1 expression by siRNA transfection similarly increased both filopodia formation and invasiveness of glioma cells as observed in Boyden chamber assay. MiR-5096 also promotes the release of extracellular vesicles by which it increases its own transfer to surrounding cells, in a Kir4.1-dependent manner in U251 but not in U87. Altogether, our results validate Kir4.1 as a miR-5096 target to promote invasion of glioblastoma cells. Our data highlight the complexity of microRNA effects and the role of K+ channels in cancer.

During the last 10 years, exosomes, which are small vesicles of 50-200 nm diameter of endosomal origin, have aroused a great interest in the scientific and clinical community for their roles in intercellular communication in almost all physiological and pathological processes. Most cells can potentially release these nanovesicles that share with the parent cell a similar lipid bilayer with transmembrane proteins and a panel of enclosed soluble proteins such as heat shock proteins and genetic material, thus acting as potential nanoshuttles of biomarkers. Exosomes surface proteins allow their targeting and capture by recipient cells, while the exosomes' content can modify the physiological state of recipient cells. Tumor derived exosomes by interacting with other cells of the tumor microenvironment modulate tumor progression, angiogenic switch, metastasis, and immune escape. Targeting tumor-derived exosomes might be an interesting approach in cancer therapy. Furthermore, because a key issue to improve cancer patients' outcome relies on earlier cancer diagnosis (metastases, as opposed to the primary tumor, are responsible for most cancer deaths) exosomes have been put forward as promising biomarker candidates for cancer diagnosis and prognosis. This review summarizes the roles of exosomes in cancer and clinical interest, focusing on the importance of exosomal heat shock proteins (HSP). The challenges of clinical translation of HSP-exosomes as therapeutic targets and biomarkers for early cancer detection are also discussed.

Background: Exosomes, via heat shock protein 70 (HSP70) expressed in their membrane, are able to interact with the toll-like receptor 2 (TLR2) on myeloid-derived suppressive cells (MDSCs), thereby activating them., Methods: We analyzed exosomes from mouse (C57Bl/6) and breast, lung, and ovarian cancer patient samples and cultured cancer cells with different approaches, including nanoparticle tracking analysis, biolayer interferometry, FACS, and electron microscopy. Data were analyzed with the Student's t and Mann-Whitney tests. All statistical tests were two-sided., Results: We showed that the A8 peptide aptamer binds to the extracellular domain of membrane HSP70 and used the aptamer to capture HSP70 exosomes from cancer patient samples. The number of HSP70 exosomes was higher in cancer patients than in healthy donors (mean, ng/mL ± SD = 3.5 ± 1.7 vs 0.17 ± 0.11, respectively, P = .004). Accordingly, all cancer cell lines examined abundantly released HSP70 exosomes, whereas "normal" cells did not. HSP70 had higher affinity for A8 than for TLR2; thus, A8 blocked HSP70/TLR2 association and the ability of tumor-derived exosomes to activate MDSCs. Treatment of tumor-bearing C57Bl/6 mice with A8 induced a decrease in the number of MDSCs in the spleen and inhibited tumor progression (n = 6 mice per group). Chemotherapeutic agents such as cisplatin or 5FU increase the amount of HSP70 exosomes, favoring the activation of MDSCs and hampering the development of an antitumor immune response. In contrast, this MDSC activation was not observed if cisplatin or 5FU was combined with A8. As a result, the antitumor effect of the drugs was strongly potentiated., Conclusions: A8 might be useful for quantifying tumor-derived exosomes and for cancer therapy through MDSC inhibition., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The human inducible heat shock protein 70 (hHsp70), which is involved in several major pathologies, including neurodegenerative disorders and cancer, is a key molecular chaperone and contributes to the proper protein folding and maintenance of a large number of protein structures. Despite its role in disease, the current structural knowledge of hHsp70 is almost exclusively based on its Escherichia coli homolog, DnaK, even though these two proteins only share ~50 % amino acid identity. For the first time, we describe a complete heterologous production and purification strategy that allowed us to obtain a large amount of soluble, full-length, and non-tagged hHsp70. The protein displayed both an ATPase and a refolding activity when combined to the human Hsp40. Multi-angle light scattering and bio-layer interferometry analyses demonstrated the ability of hHsp70 to homodimerize. The role of the C-terminal part of hHsp70 was identified and confirmed by a study of a truncated version of hHsp70 that could neither dimerize nor present refolding activity.

First discovered in 1962, heat shock proteins (HSPs) are highly studied with about 35,500 publications on the subject to date. HSPs are highly conserved, function as molecular chaperones for a large panel of "client" proteins and have strong cytoprotective properties. Induced by many different stress signals, they promote cell survival in adverse conditions. Therefore, their roles have been investigated in several conditions and pathologies where HSPs accumulate, such as in cancer. Among the diverse mammalian HSPs, some members share several features that may qualify them as cancer biomarkers. This review focuses mainly on three inducible HSPs: HSP27, HPS70, and HSP90. Our survey of recent literature highlights some recurring weaknesses in studies of the HSPs, but also identifies findings that indicate that some HSPs have potential as cancer biomarkers for successful clinical applications.

Heat shock proteins (HSPs) are necessary for cancer cell survival. We identified a mutant of HSP110 (HSP110ΔE9) in colorectal cancer showing microsatellite instability (MSI CRC), generated from an aberrantly spliced mRNA and lacking the HSP110 substrate-binding domain. This mutant was expressed at variable levels in almost all MSI CRC cell lines and primary tumors tested. HSP110ΔE9 impaired both the normal cellular localization of HSP110 and its interaction with other HSPs, thus abrogating the chaperone activity and antiapoptotic function of HSP110 in a dominant-negative manner. HSP110ΔE9 overexpression caused the sensitization of cells to anticancer agents such as oxaliplatin and 5-fluorouracil, which are routinely prescribed in the adjuvant treatment of people with CRC. The survival and response to chemotherapy of subjects with MSI CRCs was associated with the tumor expression level of HSP110ΔE9. HSP110 may thus constitute a major determinant for both prognosis and treatment response in CRC.

A major endeavour in cancer chemotherapy is to develop agents that specifically target a biomolecule of interest. There are two main classes of targeting agents: small molecules and biologics. Among biologics (e.g.: antibodies), DNA, RNA but also peptide aptamers are relatively recent agents. Peptide aptamers are seldom described but represent attractive agents that can inhibit a growing panel of oncotargets including Heat Shock Proteins. Potential pitfalls and coming challenges towards successful clinical trials are presented such as optimizing the delivery of peptide aptamers thanks to Nanotechnology.

The inhibition of heat shock protein 70 (HSP70) is an emerging strategy in cancer therapy. Unfortunately, no specific inhibitors are clinically available. By yeast two-hybrid screening, we have identified multiple peptide aptamers that bind HSP70. When expressed in human tumor cells, two among these peptide aptamers-A8 and A17-which bind to the peptide-binding and the ATP-binding domains of HSP70, respectively, specifically inhibited the chaperone activity, thereby increasing the cells' sensitivity to apoptosis induced by anticancer drugs. The 13-amino acid peptide from the variable region of A17 (called P17) retained the ability to specifically inhibit HSP70 and induced the regression of subcutaneous tumors in vivo after local or systemic injection. This antitumor effect was associated with an important recruitment of macrophages and T lymphocytes into the tumor bed. Altogether, these data indicate that peptide aptamers or peptides that target HSP70 may be considered as novel lead compounds for cancer therapy., (© 2011 AACR.)