Integrating deep learning for post-translational modifications crosstalk on Hsp90 and drug binding

The aim of this study was to investigate histone deacetylase 6 (HDAC6) modifies the heat shock protein 90 (HSP90) and heat shock transcription factor 1 (HSF1) affect the levels of pathological markers such as Aβ oligomers (Aβo) and Tau phosphorylation (p-Tau) in APP/PS1 double transgenic mice hippocampal tissues or HT22 neurons as well as the changes in cognitive behavioral functions of mice. (1) APP/PS1 transgenic mice (6 months old, 25 ~ 30g) were randomly assigned to 5 experimental groups, C57BL/6J mice (6 months old, 25 ~ 30g) were used as 4 control groups, with 8 mice in each group. All mice underwent intracerebroventricular (i.c.v.) cannulation, and the experimental groups were administered with normal saline (APP + NS group), HDAC6 agonist tubastatin A hydrochloride (TSA) (APP + TSA group) or HDAC6 agonist theophylline (Theo) (APP + Theo group), HSP90 inhibitor Ganetespib (Gane) (APP + Gane group), or a combination of pre-injected Gane by TSA (APP + Gane + TSA group); the control group received i.c.v. injections of Gane (Gane group), TSA (TSA group), Theo (Theo group) or NS (NS group), respectively. (2) Mouse hippocampal neurons HT22 were randomly divided into a control group (Control) and an Aβ1-42 intervention group (Aβ). Within the Aβ group, further divisions were made for knockdown HSP90 (Aβ + siHSP90 group), overexpression HSP90 (Aβ + OE-HSP90 group), knockdown HSF1(Aβ + siHSF1 group) and knockdown HSF1 followed by overexpression HSP90 (Aβ + siHSF1 + OE-HSP90 group), resulting in a total of 6 groups. Morris water maze test was used to evaluate the cognitive behavior of the mice. Western blot and immunohistochemistry or immunofluorescence were performed to detect the levels of HDAC6, HSP90, HSF1, Aβ1-42, Tau protein, and p-Tau in the hippocampal tissue or HT22 cells. qRT-PCR was used to measure the levels of hdac6, hsp90, and hsf1 mRNA in the hippocampus or nerve cells. (1) The levels of HDAC6, Aβ1-42 and p-Tau were elevated, while HSP90 and HSF1 were decreased in the hippocampal tissue of APP/PS1 transgenic mice (all P < 0.01). Inhibiting HDAC6 upregulated the expressions of HSP90 and HSF1 in the hippocampal tissue of APP/PS1 mice, while decreasing the levels of Aβ1-42 and p-Tau as well as improving the spatial cognitive behavior in mice (P < 0.05 or P < 0.01). The opposite effects were observed upon HDAC6 activation. However, inhibiting HSP90 reduced the expression of HSF1 (P < 0.01) and increased the levels of Aβ1-42 and p-Tau (P < 0.05 or P < 0.01) but did not significantly affect the expression of HDAC6 (P > 0.05). No significant changes were observed in the aforementioned indicators in the 4 control groups (P > 0.05). (2) In the Aβ1-42 intervention group, HDAC6 and Aβ1-42, p-Tau expression levels were elevated, while HSP90 and HSF1 expressions were all decreased, and cell viability was reduced (P < 0.05 or P < 0.01). Overexpression of HSP90 upregulated HSF1 expression, decreased the levels of Aβ1-42 and p-Tau, and increased cell viability (P < 0.05 or P < 0.01). Knocking down HSP90 had the opposite effect; and knocking down HSF1 increased the levels of Aβ1-42 and p-Tau and decreased cells viability (all P < 0.01), but did not result in significant changes in the expression levels of HSP90 (P > 0.05). Inhibiting HDAC6 can upregulate the expressions of HSP90 and HSF1 but reduce the levels of Aβ1-42 and p-Tau in the hippocampus of APP/PS1 mice and improvement of cognitive behavioral function in mice; Overexpression of HSP90 can increase HSF1 but decrease Aβ1-42 and p-Tau levels in the hippocampal neurons and increase cell activity. It is suggested that HDAC6 may affect the formation of Aβ oligomers and the changes in Tau protein phosphorylation levels in the hippocampus of AD transgenic mouse as well as the alterations in cognitive behavioral functions by regulating the HSP90-HSF1 pathway.

Serine is a key contributor to the generation of one-carbon units for DNA synthesis during cellular proliferation. In addition, it plays a crucial role in the production of antioxidants that prevent abnormal proliferation and stress in cancer cells. In recent studies, the relationship between cancer metabolism and the serine biosynthesis pathway has been highlighted. In this context, 3-phosphoglycerate dehydrogenase (PHGDH) is notable as a key enzyme that functions as the primary rate-limiting enzyme in the serine biosynthesis pathway, facilitating the conversion of 3-phosphoglycerate to 3-phosphohydroxypyruvate. Elevated PHGDH activity in diverse cancer cells is mediated through genetic amplification, posttranslational modification, increased transcription, and allosteric regulation. Ultimately, these characteristics allow PHGDH to not only influence the growth and progression of cancer but also play an important role in metastasis and drug resistance. Consequently, PHGDH has emerged as a crucial focal point in cancer research. In this review, the structural aspects of PHGDH and its involvement in one-carbon metabolism are investigated, and PHGDH is proposed as a potential therapeutic target in diverse cancers. By elucidating how PHGDH expression promotes cancer growth, the goal of this review is to provide insight into innovative treatment strategies. This paper aims to reveal how PHGDH inhibitors can overcome resistance mechanisms, contributing to the development of effective cancer treatments.

Poly(ADP-ribose)polymerase (PARP) is an enzyme responsible for catalyzing the addition of poly(ADP-ribose) chains onto target proteins in a process known as PARylation. This post-translational modification has been implicated in numerous cellular processes including gene expression, chromatin remodeling, apoptosis and DNA damage repair. In particular, the role of PARP activation in response to DNA damage has been intensely studied. BRCA mutated tumors are highly sensitive to DNA breaks and are therefore critically dependent on alternative repair mechanisms involving PARP. This forms the basis for a synthetic lethal therapeutic approach that has been met with much success in the clinic. BRCA mutated breast and ovarian tumors, in particular, are acutely sensitive to PARP inhibitors. Currently, it is unclear from in vitro and clinical studies to what extent PARP inhibitors can be used as chemotherapeutics to treat tumors beyond those harboring BRCA mutations. Due to the insensitivity of most solid tumors to PARP inhibitors, we have begun to study the role of the dePARylating enzyme poly(ADP-ribose)glycohydrolase (PARG), as a possible target in cancer therapy. PARG, like PARP, maintains a critical role in DNA repair and can therefore be a target in cells with defective repair mechanisms (e.g. BRCA mutant cells). We further hypothesize that targeting this factor may activate the expression of tumor suppressor genes, thus limiting cell proliferation. Thus far, we have novel data indicating that targeting PARG may indeed have therapeutic benefit. We show that PARG knockdowns of the T47D and MDA-MB-468 breast cancer cell lines have dramatically reduced rates of cellular proliferation. Gene expression analyses of the knockdown cells indicated that cell cycle related pathways may be affected. As such, we examined cell cycle profiles by BrdU and PI staining and found that PARG knockdown cells were arrested in S phase. These results are particularly striking given that these cells show resistance to PARP inhibition. Subsequent studies using the PARG inhibitor gallotannin successfully reconstituted the results obtained with the PARG knockdowns. Overall, our data illustrates that PARG has great potential as a new anti-cancer target. Citation Format: Amanda Lovato, Tiejun Zhao, Qiang Sun, Michael Witcher. Poly(ADP-ribose)glycohydrolase (PARG) is a novel therapeutic target in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4378. doi:10.1158/1538-7445.AM2013-4378

Identifying therapeutic targets in rare cancers remains challenging due to the paucity of established models to perform preclinical studies. As a proof-of-concept, we developed a patient-derived cancer cell line, CLF-PED-015-T, from a paediatric patient with a rare undifferentiated sarcoma. Here, we confirm that this cell line recapitulates the histology and harbours the majority of the somatic genetic alterations found in a metastatic lesion isolated at first relapse. We then perform pooled CRISPR-Cas9 and RNAi loss-of-function screens and a small-molecule screen focused on druggable cancer targets. Integrating these three complementary and orthogonal methods, we identify CDK4 and XPO1 as potential therapeutic targets in this cancer, which has no known alterations in these genes. These observations establish an approach that integrates new patient-derived models, functional genomics and chemical screens to facilitate the discovery of targets in rare cancers.

Characterization of the ERG-regulated Kinome in Prostate Cancer Identifies TNIK as a Potential Therapeutic Target

Whereas the androgen receptor (AR) signaling axis remains a therapeutic target in castration-resistant prostate cancer (CRPC), the emergence of AR mutations and splice variants as mechanisms underlying resistance to contemporary inhibitors of this pathway highlights the need for new therapeutic approaches to target this disease. Of significance in this regard is the considerable preclinical data, indicating that histone deacetylase (HDAC) inhibitors may have utility in the treatment of CRPC. However, the results of clinical studies using HDAC inhibitors (directed against HDAC1, 2, 3, and 8) in CRPC are equivocal, a result that some have attributed to their ability to induce an epithelial to mesenchymal transition (EMT) and neuroendocrine differentiation. We posited that it might be possible to uncouple the beneficial effects of HDAC inhibitors on AR signaling from their undesired activities by targeting specific HDACs as opposed to using the pan-inhibitor strategy that has been employed to date. The relative abilities of pan- and selective-Class I HDAC inhibitors to attenuate AR-mediated target gene expression and proliferation were assessed in several prostate cancer cell lines. Small interfering RNA (siRNA)-mediated knockdown approaches were used to confirm the importance of of HDAC 1, 2, and 3 expression in these processes. Further, the ability of each HDAC inhibitor to induce the expression of EMT markers (RNA and protein) and EMT-like phenotype(s) (migration) were also assessed. The anti-tumor efficacy of a HDAC3-selective inhibitor, RGFP966, was compared to the pan-HDAC inhibitor Suberoylanilide Hydroxamic Acid (SAHA) in the 22Rv1 xenograft model. Using genetic and pharmacological approaches we demonstrated that a useful inhibition of AR transcriptional activity, absent the induction of EMT, could be achieved by specifically inhibiting HDAC3. Significantly, we also determined that HDAC3 inhibitors blocked the activity of the constitutively active AR V7-splice variant and inhibited the growth of xenograft tumors expressing this protein. Our studies provide strong rationale for the near-term development of specific HDAC3 inhibitors for the treatment of CRPC.

This paper presents a technology for simple and non-iterative improvements of Multilayer and Deep Learning neural networks and Artificial Intelligence (AI) systems. The improvements are, in essence, shallow networks constructed on top of the existing Deep Learning architecture. Theoretical foundation of the technology is based on Stochastic Separation Theorems and the ideas of measure concentration. We show that, subject to mild technical assumptions on statistical properties of internal signals in Deep Learning AI, with probability close to one the technology enables instantaneous “learning away” of spurious and systematic errors. The method is illustrated with numerical examples.

The role of histone deacetylases (HDAC) and the potential of these enzymes as therapeutic targets for cancer, neurodegenerative diseases and a number of other disorders is an area of rapidly expanding investigation. There are 18 HDACs in humans. These enzymes are not redundant in function. Eleven of the HDACs are zinc dependent, classified on the basis of homology to yeast HDACs: Class I includes HDACs 1, 2, 3, and 8; Class IIA includes HDACs 4, 5, 7, and 9; Class IIB, HDACs 6 and 10; and Class IV, HDAC 11. Class III HDACs, sirtuins 1-7, have an absolute requirement for NAD(+), are not zinc dependent and generally not inhibited by compounds that inhibit zinc dependent deacetylases. In addition to histones, HDACs have many nonhistone protein substrates which have a role in regulation of gene expression, cell proliferation, cell migration, cell death, and angiogenesis. HDAC inhibitors (HDACi) have been discovered of different chemical structure. HDACi cause accumulation of acetylated forms of proteins which can alter their structure and function. HDACi can induce different phenotypes in various transformed cells, including growth arrest, apoptosis, reactive oxygen species facilitated cell death and mitotic cell death. Normal cells are relatively resistant to HDACi induced cell death. Several HDACi are in various stages of development, including clinical trials as monotherapy and in combination with other anti-cancer drugs and radiation. The first HDACi approved by the FDA for cancer therapy is suberoylanilide hydroxamic acid (SAHA, vorinostat, Zolinza), approved for treatment of cutaneous T-cell lymphoma.

Although cancer is among the most common causes of death worldwide, successful treatment options for most cancer entities remain elusive, raising the need for novel therapies. One attractive target of current drug candidates is the stress-inducible heat shock protein 90 (HSP90) chaperone machinery. Its normal chaperoning function is subverted in tumors to protect numerous mutated and overexpressed proteins from misfolding and degradation. Hence, it is playing a central role in oncogenic signaling. The addiction of cancer cells to the HSP90 chaperone machinery provides opportunities for targeting the stability of HSP90-dependent oncoproteins (clients). To dissect the importance of the HSP90 chaperone machinery in tumor progression, we investigated two HSP90-stabilized proteins: the macrophage migration inhibitory factor (MIF) in colorectal cancer (CRC) and mutant p53 (mutp53) in pancreatic ductal adenocarcinoma (PDAC). Both proteins were shown to be elevated in cancer cells via the HSP90 chaperone machinery, correlating with worse prognosis for cancer patients. MIF is a pro-inflammatory cytokine which is known to promote tumor progression in various cancer entities. Indeed, we demonstrate that loss of HSP90-stabilized MIF in CRC results in reduced tumor growth. This effect was accompanied by decreased macrophage recruitment and angiogenesis in established CRC tumors. Our data suggest that MIF acts via the CD74/MAPK axis and is indeed a cancer-relevant HSP90 client in CRC. The tumor suppressor p53 (p53) is mutated in approximately 50% of all human cancers. We found that the mutp53R248W variant is highly stabilized by the HSP90 chaperone machinery in pancreatic ductal adenocarcinoma (PDAC) cells. Furthermore, we identified a unique gain-of-function role of this p53R248W mutant on cell migration. Mechanistically, mutp53R248W specifically interacts with the phosphorylated transcription factor STAT3 and thus contributes to the aggressiveness of pancreatic cancer. Our results further corroborate HSP90 as an attractive target to counteract tumor development, and we identified two HSP90 clients as cancer drivers, outlining additional target structures for cancer therapy.

Inhibition of RAC1 activity in cancer associated fibroblasts favours breast tumour development through IL-1β upregulation.

Pancreatic adenocarcinoma (PAAD) is a highly aggressive cancer with a poor prognosis, reliable markers are urgently needed for early detection and prognosis evaluation. SAPCD2, a cell cycle related gene, has been implicated in tumorigenesis and proposed as a potential therapeutic target in cancer. However, no comprehensive study has explored its expression and regulation, discussed its role in tumor prognosis and immune modulation, along with therapy response in pan-cancer until now. SAPCD2 expression was analyzed using data from The Cancer Genome Atlas database (TCGA) and Human Protein Atlas (HPA) database. Genetic and epigenetic alterations of SAPCD2 and the immune microenvironment were explored via NCBI, TIMER2 and cBioPortal platforms. Western blot analysis and immunohistochemistry (IHC) were performed to check SAPCD2 protein expression in PAAD cells and tissues. Cell counting kit 8 (CCK8), flow cytometry, and transwell experiments were used to evaluate the role of SAPCD2 in PAAD cell lines. Our study found that SAPCD2 is notably upregulated in various cancers, especially early-stage digestive cancers, and is linked to poor survival in most cancers like PAAD and LIHC. Gene amplification and promoter DNA hypomethylation appear to drive this upregulation. Additionally, SAPCD2 expression correlates with tumor mutation burden, microsatellite instability, and immune scores across several cancers. In PAAD, elevated SAPCD2 levels correlated with reduced immune activity, whereas in stomach cancer (STAD), its prognostic impact appeared immune-independent. In PDAC cell lines, SAPCD2 knockdown reduced proliferation and invasion, and caused reduction of G0/G1 phase. PAAD cells with high SAPCD2 expression showed increased sensitivity to DNA-PK, p38α MAPK, and Bcl-2 inhibitors. SAPCD2 serves as both a prognostic marker and a potential therapeutic target in PAAD, where its low expression may enhance responsiveness to specific drugs. These findings underscore SAPCD2's dual role in cancer progression and therapy.

MicroRNAs (miRNAs) are a class of small, noncoding RNA molecules capable of regulating gene expression translationally and/or transcriptionally. A large number of evidence have demonstrated that miRNAs have a functional role in both physiological and pathological processes by regulating the expression of their target genes. Recently, the functionalities of miRNAs in the initiation, progression, angiogenesis, metastasis, and chemoresistance of tumors have gained increasing attentions. Particularly, the alteration of miRNA profiles has been correlated with the transformation and metastasis of various cancers, including colon cancer. This paper reports the latest findings on miRNAs involved in different signaling networks leading to colon cancer metastasis, mainly focusing on miRNA profiling and their roles in PTEN/PI3K, EGFR, TGFβ, and p53 signaling pathways of metastatic colon cancer. The potential of miRNAs used as biomarkers in the diagnosis, prognosis, and therapeutic targets in colon cancer is also discussed.

e16030 Background: Heat shock protein 90 (HSP90) is a chaperone for many proteins integral development of cancer, making inhibition of HSP90 an attractive strategy for cancer treatment. However, little is known about HSP90 inhibition-mediated bladder cancer therapy. Here, we present a quantitative proteomic study that evaluates alterations in protein expression and histone post-translational modifications (PTMs) in bladder cancer in response to HSP90 inhibition. Methods: We assessed the antitumor activity of five HSP90 inhibitors in 5637 bladder cancer cell line using MTS, cell viability, and caspase 3/7 assays. We then performed a proteomic analysis of cells before and after treatment with the HSP90 inhibitors and assessed for changes. Results: We show that the five HSP90 inhibitors (AUY922, ganetespib, SNX2112, AT13387, and CUDC305) potently inhibited the proliferation of bladder cancer 5637 cells. HSP90 inhibitors also had differential effects on cell survival between bladder cancer cells and uroepithelial cells. Our proteomic study quantified 518 twofold up-regulated and 811 twofold downregulated proteins common to both AUY922 and ganetespib treatment (P &lt; 0.05). The subsequent bioinformatic analysis revealed that those differentially expressed proteins were involved in chromatin modifications and cell death-associated pathways. Further, quantitative proteome studies identified 14 different types of PTMs with 93 marks on the core histones, including 34 novel histone marks of butyrylation, citrullination, 2-hydroxyisobutyrylation, propionylation, and succinylation in AUY922- and ganetespib-treated 5637 cells, suggesting that HSP90 inhibitors may exert multiple cytotoxic actions in bladder cancer cells by inhibiting HSP90 activity or altering the structure of chromatin. Conclusions: This study outlines the association between proteomic changes and histone PTMs in response to HSP90 inhibitor treatment in bladder cancer cells, and supports the notion that HSP90 inhibitors both as single agents and in combination might provide new therapeutic options for bladder cancer treatment and thus warrant further preclinical exploration.

Heat shock proteins are overexpressed in many human malignancies. The role of heat shock proteins as a therapeutic target in cancer as well as their association with drug resistance were widely documented. The aim of this study was to evaluate the concentration of IgG class HSP27 and HSP60 antibodies in serum of patients with endometrial and cervical cancer, as well as to analyse the variability of concentrations of the examined antibodies depending on the cancer stage. The study included 59 women with adenocarcinoma of the endometrium and 36 women with cervical cancer, the control group consisted of 54 healthy women. The concentrations of IgG class antibodies against the tested heat shock proteins were determined by an immunoenzymatic assay (ELISA) using commercial assays. In both endometrial and cervical cancer, the serum concentration of IgG anti-HSP27 antibody was significantly higher than in the healthy control group. The concentration of IgG anti-HSP60 antibody in endometrial cancer, cervical cancer and healthy control was similar. The median IgG anti-HSP27 antibody serum concentration of endometrial cancer patients was not correlated with FIGO-stage. In cervical cancer inverse correlation between concentration of this antibody and FIGO stage was observed. The median IgG anti-HSP60 antibody concentration in serum of endometrial cancer patients was lower in FIGO stage I and II compared to FIGO stage IV and in FIGO stage IA compared to FIGO stage IB. Concentrations of examined antibodies correlated positively with each other, both in the group of women with cancer and in the group of healthy women. The strongest correlations were found in the group of patients with endometrial cancer. Concentration of anti-HSP27 antibody could help in detection of cervical and endometrial cancer. We need to look for the cut-off point in large cohort studies. Anti-HSP27 and anti-HSP60 antibodies should be further evaluated for their potential usage as biomarkers in cervical and endometrial cancer as they shown some correlation with stage of disease.

BackgroundLysine carbamylation is a biomarker of rheumatoid arthritis and kidney diseases. However, its cellular function is understudied due to the lack of tools for systematic analysis of this post-translational modification (PTM).MethodsWe adapted a method to analyze carbamylated peptides by co-affinity purification with acetylated peptides based on the cross-reactivity of anti-acetyllysine antibodies. We also performed immobilized-metal affinity chromatography to enrich for phosphopeptides, which allowed us to obtain multi-PTM information from the same samples.ResultsBy testing the pipeline with RAW 264.7 macrophages treated with bacterial lipopolysaccharide, 7,299, 8,923 and 47,637 acetylated, carbamylated, and phosphorylated peptides were identified, respectively. Our analysis showed that carbamylation occurs on proteins from a variety of functions on sites with similar as well as distinct motifs compared to acetylation. To investigate possible PTM crosstalk, we integrated the carbamylation data with acetylation and phosphorylation data, leading to the identification 1,183 proteins that were modified by all 3 PTMs. Among these proteins, 54 had all 3 PTMs regulated by lipopolysaccharide and were enriched in immune signaling pathways, and in particular, the ubiquitin-proteasome pathway. We found that carbamylation of linear diubiquitin blocks the activity of the anti-inflammatory deubiquitinase OTULIN.ConclusionsOverall, our data show that anti-acetyllysine antibodies can be used for effective enrichment of carbamylated peptides. Moreover, carbamylation may play a role in PTM crosstalk with acetylation and phosphorylation, and that it is involved in regulating ubiquitination in vitro.Graphical 9DSWmM7hoJm8b3LaxSLrsCVideo