High Oestrogen receptor alpha expression correlates with adverse prognosis and promotes metastasis in colorectal cancer.

In normal colon tissue, oestrogen receptor alpha (ERα) is expressed at low levels, while oestrogen receptor beta (ERß) is considered the dominant subtype. However, in colon carcinomas, the ERα/ß ratio is often increased, an observation that prompted us to further investigate ERα's role in colorectal cancer (CRC). Here, we assessed ERα nuclear expression in 351 CRC patients. Among them, 119 exhibited positive ERα nuclear expression, which was significantly higher in cancer tissues than in matched normal tissues. Importantly, patients with positive nuclear ERα expression had a poor prognosis. Furthermore, positive ERα expression correlated with increased levels of the G-protein coupled cysteinyl leukotriene receptor 1 (CysLT1R) and nuclear ß-catenin, both known tumour promoters. In mouse models, ERα expression was decreased in Cysltr1-/- CAC (colitis-associated colon cancer) mice but increased in ApcMin/+ mice with wild-type Cysltr1. In cell experiments, an ERα-specific agonist (PPT) increased cell survival via WNT/ß-catenin signalling. ERα activation also promoted metastasis in a zebrafish xenograft model by affecting the tight junction proteins ZO-1 and Occludin. Pharmacological blockade or siRNA silencing of ERα limited cell survival and metastasis while restoring tight junction protein expression. In conclusion, these findings highlight the potential of ERα as a prognostic marker for CRC and its role in metastasis.

Ciclopirox ethanolamine preserves the immature state of human HSCs by mediating intracellular iron content.

Culture conditions in which hematopoietic stem cells (HSCs) can be expanded for clinical benefit are highly sought after. To elucidate regulatory mechanisms governing the maintenance and propagation of human HSCs ex vivo, we screened libraries of annotated small molecules in human cord blood cells using an optimized assay for detection of functional HSCs during culture. We found that the antifungal agent ciclopirox ethanolamine (CPX) selectively supported immature CD34+CD90+ cells during culture and enhanced their long-term in vivo repopulation capacity. Purified HSCs treated with CPX showed a reduced cell division rate and an enrichment of HSC-specific gene expression patterns. Mechanistically, we found that the HSC stimulating effect of CPX was directly mediated by chelation of the intracellular iron pool, which in turn affected iron-dependent proteins and enzymes mediating cellular metabolism and respiration. Our findings unveil a significant impact of iron homeostasis in regulation of human HSCs, with important implications for both basic HSC biology and clinical hematology.

In situ assembly of bioresorbable organic bioelectronics in the brain.

Bioelectronics can potentially complement classical therapies in nonchronic treatments, such as immunotherapy and cancer. In addition to functionality, minimally invasive implantation methods and bioresorbable materials are central to nonchronic treatments. The latter avoids the need for surgical removal after disease relief. Self-organizing substrate-free organic electrodes meet these criteria and integrate seamlessly into dynamic biological systems in ways difficult for classical rigid solid-state electronics. Here we place bioresorbable electrodes with a brain-matched shear modulus-made from water-dispersed nanoparticles in the brain-in the targeted area using a capillary thinner than a human hair. Thereafter, we show that an optional auxiliary module grows dendrites from the installed conductive structure to seamlessly embed neurons and modify the electrode's volume properties. We demonstrate that these soft electrodes set off a controlled cellular response in the brain when relaying external stimuli and that the biocompatible materials show no tissue damage after bioresorption. These findings encourage further investigation of temporary organic bioelectronics for nonchronic treatments assembled in vivo.

Glioblastoma CD105+ cells define a SOX2- cancer stem cell-like subpopulation in the pre-invasive niche.

Glioblastoma (GBM) is the most common and most aggressive primary brain tumor in adults. Glioma stem like cells (GSC) represent the highest cellular hierarchy in GBM and have a determining role in tumor growth, recurrence and patient prognosis. However, a better definition of GSC subpopulations, especially at the surgical resection margin, is warranted for improved oncological treatment options. The present study interrogated cells expressing CD105 (CD105+) specifically within the tumor front and the pre-invasive niche as a potential GSC subpopulation. GBM primary cell lines were generated from patients (n = 18) and CD105+ cells were isolated and assessed for stem-like characteristics. In vitro, CD105+ cells proliferated and enriched in serum-containing medium but not in serum-free conditions. CD105+ cells were characterized by Nestin+, Vimentin+ and SOX2-, clearly distinguishing them from SOX2+ GCS. GBM CD105+ cells differentiated into osteocytes and adipocytes but not chondrocytes. Exome sequencing revealed that GBM CD105+ cells matched 83% of somatic mutations in the Cancer cell line encyclopedia, indicating a malignant phenotype and in vivo xenotransplantation assays verified their tumorigenic potential. Cytokine assays showed that immunosuppressive and protumorigenic cytokines such as IL6, IL8, CCL2, CXCL-1 were produced by CD105+ cells. Finally, screening for 88 clinical drugs revealed that GBM CD105+ cells are resistant to most chemotherapeutics except Doxorubicin, Idarubicin, Fludarabine and ABT-751. Our study provides a rationale for targeting tumoral CD105+ cells in order to reshape the tumor microenvironment and block GBM progression.

High PGD2 receptor 2 levels are associated with poor prognosis in colorectal cancer patients and induce VEGF expression in colon cancer cells and migration in a zebrafish xenograft model.

BACKGROUND: Despite intense research, the prognosis for patients with advanced colorectal cancer (CRC) remains poor. The prostaglandin D2 receptors DP1 and DP2 are explored here as potential therapeutic targets for advanced CRC. METHODS: A CRC cohort was analysed to determine whether DP1 and DP2 receptor expression correlates with patient survival. Four colon cancer cell lines and a zebrafish metastasis model were used to explore how DP1/DP2 receptor expression correlates with CRC progression. RESULTS: Analysis of the clinical CRC cohort revealed high DP2 expression in tumour tissue, whereas DP1 expression was low. High DP2 expression negatively correlated with overall survival. Other pathological indicators, such as TNM stage and metastasis, positively correlated with DP2 but not DP1 expression. In accordance, the in vitro results showed high DP2 expression in four CC-cell lines, but only one expressed DP1. DP2 stimulation resulted in increased proliferation, p-ERK1/2 and VEGF expression/secretion. DP2-stimulated cells exhibited increased migration in the zebrafish metastasis model. CONCLUSION: Our results support DP2 receptor expression and signalling as a therapeutic target in CRC progression based on its expression in CRC tissue correlating with poor patient survival and that it triggers proliferation, p-ERK1/2 and VEGF expression and release and increased metastatic activity in CC-cells.

Tumour suppressor 15-hydroxyprostaglandin dehydrogenase induces differentiation in colon cancer via GLI1 inhibition.

Inflammation is an established risk factor for colorectal cancer. We and others have shown that colorectal cancer patients with elevated cysteinyl leukotriene receptor 2 (CysLT2R) and 15-hydroxyprostaglandin dehydrogenase (15-PGDH) levels exhibit good prognoses. However, both CysLT2R and 15-PGDH, which act as tumour suppressors, are often suppressed in colorectal cancer. We previously reported that leukotriene C4 (LTC4)-induced differentiation in colon cancer via CysLT2R signalling. Here, we investigated the involvement of Hedgehog (Hh)-GLI1 signalling, which is often hyperactivated in colorectal cancer. We found that the majority of colorectal cancer patients had high-GLI1 expression, which was negatively correlated with CysLT2R, 15-PGDH, and Mucin-2 and overall survival compared with the low-GLI1 group. LTC4-induced 15-PGDH downregulated both the mRNA and protein expression of GLI1 in a protein kinase A (PKA)-dependent manner. Interestingly, the LTC4-induced increase in differentiation markers and reduction in Wnt targets remained unaltered in GLI1-knockdown cells. The restoration of GLI1 in 15-PGDH-knockdown cells did not ameliorate the LTC4-induced effects, indicating the importance of both 15-PGDH and GLI1. LTC4-mediated reduction in the DCLK1 and LGR5 stemness markers in colonospheres was abolished in cells lacking 15-PGDH or GLI1. Both DCLK1 and LGR5 were highly increased in tumour tissue compared with the matched controls. Reduced Mucin-2 levels were observed both in zebrafish xenografts with GLI1-knockdown cells and in the cysltr2-/- colitis-associated colon cancer (CAC) mouse model. Furthermore, GLI1 expression was positively correlated with stemness and negatively correlated with differentiation in CRC patients when comparing tumour and mucosal tissues. In conclusion, restoring 15-PGDH expression via CysLT2R activation might benefit colorectal cancer patients.

Tumour-suppressive effect of oestrogen receptor ß in colorectal cancer patients, colon cancer cells, and a zebrafish model.

Oestrogen receptor ß (ERß) has been suggested to have anti-proliferative and anti-tumour effects in breast and prostate cancer cells, but other studies have indicated its tumour-promoting effects. Understanding the complex effects of this receptor in different contexts requires further study. We reported that high ERß expression is independently associated with improved prognosis in female colorectal cancer (CRC) patients. Herein, we investigated the possible anti-tumour effect of ERß and its selective agonist. CRC patients with high ERß expression had significantly higher levels of membrane-associated ß-catenin, cysteinyl leukotriene receptor 2 (CysLT2 R), and 15-hydroxyprostaglandin dehydrogenase (15-PGDH), which have anti-tumour effects, but lower levels of nuclear ß-catenin, cysteinyl leukotriene receptor 1 (CysLT1 R), and cyclooxygenase-2 (COX-2), which have tumour-promoting effects. These interesting findings were further supported by two different publicly available CRC mRNA datasets that showed a significant positive correlation between ERß expression and 15-PGDH and CysLT2 R expression and a negative correlation between ERß expression and ß-catenin, CysLT1 R, and COX-2 expression. We next evaluated ERß expression in three different colon cancer mouse models; ERß expression was negatively correlated with tumourigenesis. Furthermore, treatment with the ERß-agonist ERB-041 reduced CysLT1 R, active ß-catenin, and COX-2 levels but increased phospho-ß-catenin, CysLT2 R, and 15-PGDH levels in HCT-116, Caco-2, and SW-480 colon cancer cells compared to vehicle-treated cells. Interestingly, ERB-041-treated cells showed significantly decreased migration, survival, and colonosphere formation and increased apoptotic activity, as indicated by increased CASPASE-3 and apoptotic blebs. Finally, patients with low ERß expression had significantly more distant metastasis at the time of diagnosis than patients with high ERß expression. Therefore, we studied the effects of ERB-041-treated colon cancer cells in a zebrafish xenograft model. We found significantly less distant metastasis of ERB-041-treated cells compared to vehicle-treated cells. These results further support ERß's anti-tumour role in CRC and the possible use of its agonist in CRC patients. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

A Phenotypic Screening Assay Identifies Modulators of Diamond Blackfan Anemia.

Diamond-Blackfan anemia (DBA) is a bone marrow failure syndrome caused by mutations in ribosomal protein genes. Pathogenic mechanisms are poorly understood but involve severely reduced proliferation of erythroid precursors. Because current DBA therapies are ineffective and associated with severe side effects, disease-specific therapies are urgently needed. We hypothesized that druggable molecular pathways underlying the defect can be revealed through phenotypic small-molecule screens. Accordingly, a screening assay was developed using c-kit+ fetal liver erythroid progenitors from a doxycycline-inducible DBA mouse model. The addition of doxycycline to the culture medium induces the phenotype and reduces proliferation to <10% of normal, such that rescue of proliferation can be used as a simple readout for screening. Here, we describe the assay rationale and efforts toward validation of a microtiter plate-compatible assay and its application in a pilot screen of 3871 annotated compounds. Ten hits demonstrated concentration-dependent activity, and we report a brief follow-up of one of these compounds. In conclusion, we established a robust scalable assay for screening molecules that rescue erythropoiesis in DBA.

Bortezomib prevents cytarabine resistance in MCL, which is characterized by down-regulation of dCK and up-regulation of SPIB resulting in high NF-κB activity.

BACKGROUND: The addition of high-dose cytarabine to the treatment of mantle cell lymphoma (MCL) has significantly prolonged survival of patients, but relapses are common and are normally associated with increased resistance. To elucidate the mechanisms responsible for cytarabine resistance, and to create a tool for drug discovery investigations, we established a unique and molecularly reproducible cytarabine resistant model from the Z138 MCL cell line. METHODS: Effects of different substances on cytarabine-sensitive and resistant cells were evaluated by assessment of cell proliferation using [methyl-14C]-thymidine incorporation and molecular changes were investigated by protein and gene expression analyses. RESULTS: Gene expression profiling revealed that major transcriptional changes occur during the initial phase of adaptation to cellular growth in cytarabine containing media, and only few key genes, including SPIB, are deregulated upon the later development of resistance. Resistance was shown to be mediated by down-regulation of the deoxycytidine kinase (dCK) protein, responsible for activation of nucleoside analogue prodrugs. This key event, emphasized by cross-resistance to other nucleoside analogues, did not only effect resistance but also levels of SPIB and NF-κB, as assessed through forced overexpression in resistant cells. Thus, for the first time we show that regulation of drug resistance through prevention of conversion of pro-drug into active drug are closely linked to increased proliferation and resistance to apoptosis in MCL. Using drug libraries, we identify several substances with growth reducing effect on cytarabine resistant cells. We further hypothesized that co-treatment with bortezomib could prevent resistance development. This was confirmed and show that the dCK levels are retained upon co-treatment, indicating a clinical use for bortezomib treatment in combination with cytarabine to avoid development of resistance. The possibility to predict cytarabine resistance in diagnostic samples was assessed, but analysis show that a majority of patients have moderate to high expression of dCK at diagnosis, corresponding well to the initial clinical response to cytarabine treatment. CONCLUSION: We show that cytarabine resistance potentially can be avoided or at least delayed through co-treatment with bortezomib, and that down-regulation of dCK and up-regulation of SPIB and NF-κB are the main molecular events driving cytarabine resistance development.

[Surgical quality registries: large room for improvement if they should be used for analyses of life style risk factors and the effect of preoperative initiatives on postoperative complications]. / Livsstilsfaktorer förbisedda i kirurgiska kvalitetsregister - Registren kan ännu inte användas för att utvärdera preoperativa hälsoinsatser och levnadsvanors påverkan på komplikationer.

Surgical quality registries: large room for improvement if they should be used for analyses of life style risk factors and the effect of preoperative initiatives on postoperative complications Tobacco, alcohol, malnutrition, overweight and physical inactivity increase postoperative morbidity. Preoperative intervention has been shown to improve the outcome significantly. The aim was to investigate if these lifestyle risk factors were reported in the Swedish national surgical quality registries together with preoperative interventions and postoperative complications. Altogether, 44 registers reporting on surgical procedures were identified. Preoperative documentation existed in about half of the registries, but relevant health promoting intervention was not documented. Postoperative complications were reported with varying degrees. No registry had complete reporting of preoperative lifestyle risk factors, related intervention and postoperative complications. For most registries, there is a large room for improvement if they should be used for analyses of risk factors and the effect of implementation of preoperative initiatives on postoperative complications.

Small molecules increase direct neural conversion of human fibroblasts.

The generation of human induced neurons (hiNs) via exogenous delivery of neural transcription factors represents a novel technique to obtain disease and patient specific neurons. These cells have the potential to be used for disease modeling, diagnostics and drug screening, and also to be further developed for brain repair. In the present study, we utilized hiNs to develop an unbiased screening assay for small molecules that increase the conversion efficiency. Using this assay, we screened 307 compounds from five annotated libraries and identified six compounds that were very potent in potentiating the reprogramming process. When combined in an optimal combination and dose, these compounds increased the reprogramming efficiency of human fibroblasts more than 6-fold. Global gene expression and CellNet analysis at different timepoints during the reprogramming process revealed that neuron-specific genes and gene regulatory networks (GRNs) became progressively more activated while converting cells shut down fibroblast-specific GRNs. Further bioinformatics analysis revealed that the addition of the six compound resulted in the accelerated upregulation of a subset of neuronal genes, and also increased expression of genes associated with transcriptional activity and mediation of cellular stress response.

Identification of V-ATPase as a molecular sensor of SOX11-levels and potential therapeutic target for mantle cell lymphoma.

BACKGROUND: Mantle cell lymphoma (MCL) is an aggressive disease with short median survival. Molecularly, MCL is defined by the t(11;14) translocation leading to overexpression of the CCND1 gene. However, recent data show that the neural transcription factor SOX11 is a disease defining antigen and several involved signaling pathways have been pin-pointed, among others the Wnt/ß-catenin pathway that is of importance for proliferation in MCL. Therefore, we evaluated a compound library focused on the Wnt pathway with the aim of identifying Wnt-related targets that regulate growth and survival in MCL, with particular focus on SOX11-dependent growth regulation. METHODS: An inducible SOX11 knock-down system was used to functionally screen a library of compounds (n = 75) targeting the Wnt signaling pathway. A functionally interesting target, vacuolar-type H(+)-ATPase (V-ATPase), was further evaluated by western blot, siRNA-mediated gene silencing, immunofluorescence, and flow cytometry. RESULTS: We show that 15 out of 75 compounds targeting the Wnt pathway reduce proliferation in all three MCL cell lines tested. Furthermore, three substances targeting two different targets (V-ATPase and Dkk1) showed SOX11-dependent activity. Further validation analyses were focused on V-ATPase and showed that two independent V-ATPase inhibitors (bafilomycin A1 and concanamycin A) are sensitive to SOX11 levels, causing reduced anti-proliferative response in SOX11 low cells. We further show, using fluorescence imaging and flow cytometry, that V-ATPase is mainly localized to the plasma membrane in primary and MCL cell lines. CONCLUSIONS: We show that SOX11 status affect V-ATPase dependent pathways, and thus may be involved in regulating pH in intracellular and extracellular compartments. The plasma membrane localization of V-ATPase indicates that pH regulation of the immediate extracellular compartment may be of importance for receptor functionality and potentially invasiveness in vivo.

Estrogen Receptor Beta Selective Agonists as Agents to Treat Chemotherapeutic-Induced Neuropathic Pain.

Chemotherapy-induced neuropathic pain (CINP) remains a major unmet medical need. Estrogen receptor beta (ERß)-selective agonists represent a novel strategy for treating CINP because they are neuroprotective and may also have anticancer activity. We confirmed that ERß-selective agonists have antiallodynic effects in the spinal nerve ligation model of neuropathic pain. We then showed that structurally diverse ERß-selective agonists also relieved allodynia in CINP caused by taxol, oxaliplatin, and vincristine. These effects were receptor subtype specific and mediated by ERß receptors as ERα-selective and nonselective estrogen agonists were inactive, a mixture of an ERß and ERα agonist was inactive, and ERß-selective antagonists blocked the effects of the ERß-selective agonists. The efficacy and potency of ERß-agonists was greater in male rats than females. To address the possibility that AC-186 might stimulate proliferation of cancers, rendering it unsuitable for treating CINP, we evaluated proliferative effects of AC-186 on prostate cancer cells and found it inhibited growth (LNCaP cells) or had no effect (PC3 cells) on these cells. Thus, ERß-selective agonists exhibit potential for treating CINP.

Therapeutic Targeting of Nuclear γ-Tubulin in RB1-Negative Tumors.

UNLABELLED: In addition to its cytosolic function, γ-tubulin is a chromatin-associated protein. Reduced levels of nuclear γ-tubulin increase the activity of E2 promoter-binding factors (E2F) and raise the levels of retinoblastoma (RB1) tumor suppressor protein. In tumor cells lacking RB1 expression, decreased γ-tubulin levels induce cell death. Consequently, impairment of the nuclear activity of γ-tubulin has been suggested as a strategy for targeted chemotherapy of RB1-deficient tumors; thus, tubulin inhibitors were tested to identify compounds that interfere with γ-tubulin. Interestingly, citral increased E2F activity but impaired microtubule dynamics while citral analogues, such citral dimethyl acetal (CDA), increased E2F activity without affecting microtubules. The cytotoxic effect of CDA on tumor cells was attenuated by increased expression of either RB1 or γ-tubulin, and increased by reduced levels of either RB1 or γ-tubulin. Mechanistic study, in silico and in vitro, demonstrated that CDA prevents GTP binding to γ-tubulin and suggested that the FDA-approved drug dimethyl fumarate is also a γ-tubulin inhibitor. Finally, in vivo growth of xenograft tumors carrying defects in the RB1 signaling pathway were inhibited by CDA treatment. These results demonstrate that inhibition of γ-tubulin has the potential to specifically target tumor cells and may aid in the design of safer and more efficient chemotherapeutic regimes. IMPLICATIONS: The in vivo antitumorigenic activity of γ-tubulin inhibitors paves the way for the development of a novel broad range targeted anticancer therapy that causes fewer side effects.

Synthesis and biological evaluation of second-generation tropanol-based androgen receptor modulators.

To circumvent antiandrogen resistance in prostate cancer, antiandrogens effective for both the androgen receptor (AR) and AR mutants are required. The AR antagonists in this study originate from previous findings, which showed that subtle differences in substitution pattern lead to a conformational change that alters the ligand activity, rendering an agonist to an antagonist. We have identified small yet potent tropanol-based ligands possessing significant antiandrogenic activity with both wild-type AR and the two most common AR ligand binding domain (LBD) mutants.

Apicidin sensitizes pancreatic cancer cells to gemcitabine by epigenetically regulating MUC4 expression.

BACKGROUND/AIM: Mucin 4 (MUC4) has been linked to resistance to gemcitabine in pancreatic cancer cells. The aim of the present study was to assess whether epigenetic control of MUC4 expression can sensitize pancreatic cancer cells to gemcitabine treatment. MATERIALS AND METHODS: A 76-member combined epigenetics and phosphatase small-molecule inhibitor library was screened for anti-proliferative activity against the MUC4(+) gemcitabine-resistant pancreatic cancer cell line Capan-1, followed by high-content screening of protein expression. RESULTS: Apicidin, a histone deacetylase inhibitor, showed the greatest anti-proliferative activity with a lethal dose 50 (LD50) value of 5.17 µM. Apicidin significantly reduced the expression of MUC4 and its transcription factor hepatocyte nuclear factor 4α. Combined treatment with a sub-therapeutic concentration of apicidin and gemcitabine synergistically inhibited growth of Capan-1 cells. CONCLUSION: Apicidin appears to be a novel anti-proliferative agent against pancreatic cancer cells that may reverse chemoresistance by epigenetically regulating MUC4 expression.

Impact of estrogen receptor beta activation on functional recovery after experimental stroke.

Acute treatment with 17ß-estradiol provides effective neuroprotection during the first days after acute brain injury, however, effects of chronic activation of estrogen receptor beta (ERß) on recovery of function after experimental stroke have not been investigated. The present study, therefore, was conducted to test if delayed treatment with the specific ERß ligand 4-(1-phenyl-cyclohexyl)-phenol (AC-131) improves recovery of lost neurological function after permanent focal stroke induced by photothrombosis in adult Sprague-Dawley rats. Treatment was initiated on day 2 after photothrombosis and AC-131 (1, 10, and 50 mg/kg) was administered by daily subcutaneous injections for 14 days. On day 2, 4, 6, 8, 11, 14, and 17 after photothrombosis, functional deficits were assessed by the paw placement test, a standardized grip strength test and an adhesive removal test. Daily treatment with AC-131 significantly improved test scores in all three behavioral tests. Importantly, improved function was not associated with a decrease in infarct volume on day 17 after stroke onset. Our results suggest that increased activity of the ERß is involved in mechanisms of stroke recovery.

Low dose bexarotene treatment rescues dopamine neurons and restores behavioral function in models of Parkinson's disease.

Nurr1 is a nuclear hormone receptor (NucHR) strongly implicated in the growth, maintenance, and survival of dopaminergic neurons. Nurr1 may be unable to bind ligands directly, but it forms heterodimers with other NucHRs that do. Using bioluminescence resonance energy transfer (BRET) assays to directly monitor interactions of Nurr1 with other NucHRs, we found the cancer drug bexarotene (Targretin, also LGD1069) displayed biased interactions with Nurr1-RXR heterodimers compared with RXR-RXR homodimers. Remarkably, at doses up to 100-fold lower than those effective in rodent cancer models, bexarotene rescued dopamine neurons and reversed behavioral deficits in 6-hydroxydopamine (6-OHDA) lesioned rats. Compared to the high doses used in cancer therapy, low doses of bexarotene have significantly milder side effects including a reduced increase in plasma triglycerides and less suppression of thyroid function. On the basis of extrapolations from rat to human doses, we hypothesize that low oral doses of bexarotene may provide an effective and tolerated therapy for Parkinson's disease (PD).