Identification of the DEAD box RNA helicase DDX3 as a therapeutic target in colorectal cancer

Identifying druggable targets in the Wnt-signaling pathway can optimize colorectal cancer treatment. Recent studies have identified a member of the RNA helicase family DDX3 (DDX3X) as a multilevel activator of Wnt signaling in cells without activating mutations in the Wnt-signaling pathway. In this study, we evaluated whether DDX3 plays a role in the constitutively active Wnt pathway that drives colorectal cancer.We determined DDX3 expression levels in 303 colorectal cancers by immunohistochemistry. 39% of tumors overexpressed DDX3. High cytoplasmic DDX3 expression correlated with nuclear β-catenin expression, a marker of activated Wnt signaling. Functionally, we validated this finding in vitro and found that inhibition of DDX3 with siRNA resulted in reduced TCF4-reporter activity and lowered the mRNA expression levels of downstream TCF4-regulated genes. In addition, DDX3 knockdown in colorectal cancer cell lines reduced proliferation and caused a G1 arrest, supporting a potential oncogenic role of DDX3 in colorectal cancer.RK-33 is a small molecule inhibitor designed to bind to the ATP-binding site of DDX3. Treatment of colorectal cancer cell lines and patient-derived 3D cultures with RK-33 inhibited growth and promoted cell death with IC50 values ranging from 2.5 to 8 μM. The highest RK-33 sensitivity was observed in tumors with wild-type APC-status and a mutation in CTNNB1.Based on these results, we conclude that DDX3 has an oncogenic role in colorectal cancer. Inhibition of DDX3 with the small molecule inhibitor RK-33 causes inhibition of Wnt signaling and may therefore be a promising future treatment strategy for a subset of colorectal cancers.     

Heat shock protein expression and autophagy after incomplete thermal ablation and their correlation

Objective: To establish a model of incomplete ablation in nude mice with hepatocellular carcinoma (HCC) and to evaluate heat shock protein (HSP) expression and autophagy and their correlation.

Materials and methods: In the first stage, 12 nude mice with HCC were randomly divided into two groups (n?=?6). A sham puncture operation was performed for one group, and palliative laser ablation was performed for the other group. All mice were sacrificed after 18?h, and HSP expression, autophagy, and apoptosis were assessed. In the second stage, 16 nude mice with HCC were randomly divided into two groups (n?=?8). One group was given an HSP90 inhibitor before the operation, and the other group was given dimethyl sulfoxide (DMSO) as a control. HSP expression, autophagy and apoptosis were assessed for the two groups after palliative laser ablation.

Results: In the incomplete ablation model, using nude mice with HCC, HSP90, HSP70, and HSP27 expression was up-regulated, Akt and mTOR phosphorylation was enhanced, autophagy was decreased, and apoptosis was increased. After administration of the HSP90 inhibitor, HSP90, P-Akt, and P-mTOR expression was decreased, autophagy was increased, and apoptosis was further increased.

Conclusion: Autophagy was decreased in the incomplete ablation model and might be inversely correlated with HSP expression. It is suggested that the HSP90/Akt/mTOR pathway is involved in signal transmission between autophagy and HSPs.     

Therapeutic efficacy of atezolizumab plus bevacizumab for hepatocellular carcinoma with WNT/β-catenin signal activation

Atezolizumab plus bevacizumab therapy has high response rates in patients with advanced hepatocellular carcinoma (HCC). It has been reported that HCC with immune exclusion associated with the signal activation of WNT/β-catenin is resistant to immune checkpoint inhibitors; however, to the best of our knowledge, the effectiveness of atezolizumab plus bevacizumab for HCC with WNT/β-catenin signal activation has not been reported. The present study aimed to analyze the efficacy of atezolizumab plus bevacizumab for HCC with WNT/β-catenin signal activation. A total of 24 patients who underwent liver tumor biopsy for HCC were classified into WNT/β-catenin signal activation and inactivation groups according to the expression levels of β-catenin and glutamine synthetase, which are indicative of WNT/β-catenin signal activation. The differences in the clinical responses to treatment between the groups were analyzed. A total of 15 patients had HCC with WNT/β-catenin signal activation, whereas 9 patients had HCC with WNT/β-catenin signal inactivation. There were no significant differences between both groups regarding objective responses (P=0.519) and disease control (P=0.586). In the WNT/β-catenin signal activation group, the median progression-free survival rate was 6.9 months compared with 6.2 months in the WNT/β-catenin signal inactivation group (P=0.674). Although a small number of patients was included in the present study, the present findings suggested that the efficacy of atezolizumab plus bevacizumab might be unaffected by WNT/β-catenin signal activation.     

TM2D1 contributes the epithelial-mesenchymal transition of hepatocellular carcinoma via modulating AKT/β-catenin axis

Various epidemiology studies showed the correlation between Alzheimer’s disease (AD) and low incidence of cancer. However, the etiology underlying etiology of AD-related carcinogenesis remains largely elusive. Our study focused on characterizing the role of TM2D1 (TM2 domain containing 1) in hepatocellular carcinoma. TM2D1 is also known as β-amyloid peptide binding protein and is critical to the pathogenesis of AD. We found that TM2D1 is increasingly expressed in HCC tumors relative to the peritumoral tissues of the matched tumors and high TM2D1 expression predicts unfavorable clinical outcomes. TM2D1 overexpression induced HCC cell proliferation, migration and invasion, which was related to the epithelial-mesenchymal transition (EMT) observed in these cells. Conversely, TM2D1 depletion led to opposite phenotype in HCC. Mechanistically, we found that TM2D1 promoted Akt and β-catenin hyper-activation, which corresponded with molecular marker change in EMT signaling pathway. Taken together, our results indicated that TM2D1 played an important role in the EMT process in HCC cells by activating AKT and β-catenin signaling and may become a promising therapeutic target in HCC.     

Utility and specificity of plasma heat shock protein 90 alpha,CEA, and CA199 as the diagnostic test in colorectal cancer liver metastasis

BackgroundPlasma heat shock protein 90 alpha (Hsp90α) has been suggested as a novel biomarker for the diagnosis and prognosis of cancer. Carcinoembryonic antigen (CEA) and carbohydrate antigen199 (CA199) are traditional tumor biomarkers for colorectal cancer (CRC). Previous studies have shown that Hsp90α and the combination of Hsp90α and CEA are optimal biomarkers for CRC at an early stage. However, research on the use of Hsp90α alone or in combination with CEA and/or CA199 in diagnosing CRC development, particularly liver metastasis, is limited. This study sought to investigate the value of Hsp90α alone or in combination with CEA/CA199 in diagnosing CRC liver metastasis.MethodsThe clinical data of 472 CRC patients were retrospectively analyzed, which were confirmed by clinical manifestations and a histopathological examination associated with an imaging diagnosis. The levels of Hsp90α, and CEA, and CA199 were assessed by enzyme-linked immunoassays and electrochemiluminescence immunoassays. Liver metastasis was diagnosed by imaging or pathology of the liver. Logistic regression models were used to analyze associations between Hsp90α, CEA, and CA199, and liver metastasis in CRC. The areas under the curves (AUCs) were used to compare the utility of Hsp90α, CEA, and CA199 in the diagnosis of CRC liver metastasis (CRLM). Additionally, we compared the diagnostic utility of the models, including the Hsp90α plus 1 of the other serum markers, and a combination of the 3 serum makers.ResultsThe plasma levels of Hsp90α, CEA, and CA199 were positively associated with a higher risk of CRLM [odds ratios (OR) ranging from 1.36–2.72]. The AUCs of CEA, CA199, and Hsp90α for CRLM were 0.80, 0.69, and 0.55, respectively. The AUCs for the combination of Hsp90α and CEA, combination of Hsp90α and CA199, combinations of Hsp90α, CEA, and CA199 were 0.75, 0.66, 0.76, respectively. The combination of Hsp90α, CEA, and CA199 did not improve the diagnostic utility for liver metastasis in CRC.ConclusionsThe level of Hsp90α was elevated in CRC and was associated with CRLM. Thus, the Hsp90α is a potential biomarker for CRLM. CEA has the largest diagnostic utility for CRLM. Adding Hsp90α to CEA/CA199 did not improve their diagnostic utility for CRLM.     

MicroRNA-182 promotes epithelial-mesenchymal transition by targeting FOXN3 in gallbladder cancer

Increasing evidence has suggested an association between the expression profiles of microRNAs (miRs) and gallbladder cancer (GBC). Recently, miR-182 has been demonstrated to exert tumor-promoting effects. However, the biological activity and molecular mechanisms of miR-182 in GBC remain unclear. The results of the present study demonstrated that miR-182 expression was significantly upregulated in GBC tissues and cell lines (GBC-SD and SGC-996). In addition, miR-182-knockdown attenuated epithelial-mesenchymal transition (EMT) in GBC cells, as indicated by decreased cell migratory and invasive abilities, decreased vimentin expression, and increased E-cadherin expression. The activities of β-catenin and its downstream factors, Cyclin D1 and c-Myc, were also demonstrated to decrease following miR-182-knockdown. Forkhead box N3 (FOXN3) was identified as the direct target of miR-182. Overexpression of FOXN3 ameliorated EMT and the β-catenin pathway. Taken together, the results of the present study suggested that miR-182 promotes EMT in GBC cells by targeting FOXN3, which suppresses the Wnt/β-catenin pathway.     

Anti-proliferative activity of heat shock protein (Hsp) 90 inhibitors via β-catenin/TCF7L2 pathway in adult T cell leukemia cells

The aim of this study is to evaluate the effect of heat shock protein 90 (Hsp90) inhibition, and to identify molecular pathways responsible for anti-proliferative effect on adult T cell leukemia/lymphoma (ATL) cells. For Hsp90 inhibition, we used geldanamycin derivates, 17-AAG (17-allylamino-17-demethoxygeldanamycin) and 17-DMAG (17-(dimethylaminoethylamino) 17-demethoxygeldanamycin) in this study. The inhibitory concentration (IC₅₀) of 17-AAG in an ATL cell line, designated as TaY, and two HTLV-1 transformed cell lines (MT-2 and MT-4) was 300–700 nM, and that of 17-DMAG was 150–200 nM. Fresh ATL cells obtained from patients were more sensitive to both 17-AAG and 17-DMAG. Gene expression analysis of TaY cells revealed up-regulation of HSPA1A encoding Hsp70, a hallmark of Hsp90 inhibition. Genes regulating cell proliferation or anti-apoptosis (i.e. BCL2 and BIRC5), genes related to cytokines or chemokines (i.e. IL9 and CCL27), and notably TCF7L2, a down-stream effecter of β-catenin were remarkably down-regulated. Down-regulation of TCF7L2 mRNA was noted in the three cell lines and two patient specimens after Hsp90 inhibition. Hsp90 inhibitors dephosphorylate AKT, thereby, activate GSK-3β, which phosphorylates β-catenin for ubiquitination. This indicates the possibility that β-catenin/TCF7L2 pathway plays an important role in Hsp90 inhibitor-induced cell death in ATL cells and HTLV-1 transformed cells. Our results have provided new insights into the complex molecular pharmacology of Hsp90 inhibitors, and suggest that Hsp90 inhibitors might be beneficial as anti-proliferative agents in treating ATL patients.     

SIK2 represses AKT/GSK3β/β‐catenin signaling and suppresses gastric cancer by inhibiting autophagic degradation of protein phosphatases

Salt‐inducible kinase 2 (SIK2) is an important regulator in various intracellular signaling pathways related to apoptosis, tumorigenesis and metastasis. However, the involvement of SIK2 in gastric tumorigenesis and the functional linkage with gastric cancer (GC) progression remain to be defined. Here, we report that SIK2 was significantly downregulated in human GC tissues, and reduced SIK2 expression was associated with poor prognosis of patients. Overexpression of SIK2 suppressed the migration and invasion of GC cells, whereas knockdown of SIK2 enhanced cell migratory and invasive capability as well as metastatic potential. These changes in the malignant phenotype resulted from the ability of SIK2 to suppress epithelial–mesenchymal transition via inhibition of AKT/GSK3β/β‐catenin signaling. The inhibitory effect of SIK2 on AKT/GSK3β/β‐catenin signaling was mediated primarily through inactivation of AKT, due to its enhanced dephosphorylation by the upregulated protein phosphatases PHLPP2 and PP2A. The upregulation of PHLPP2 and PP2A was attributable to SIK2 phosphorylation and activation of mTORC1, which inhibited autophagic degradation of these two phosphatases. These results suggest that SIK2 acts as a tumor suppressor in GC and may serve as a novel prognostic biomarker and therapeutic target for this tumor.

Abbreviations

AMPK

AMP‐activated protein kinase

Co‐IP

co‐immunoprecipitation

EMT

epithelial–mesenchymal transition

GAPDH

glyceraldehyde‐3‐phosphate dehydrogenase

GC

gastric cancer

GEO

Gene Expression Omnibus

H&E

hematoxylin and eosin

IHC

immunohistochemistry

mTOR

mechanistic target of rapamycin

NC

negative control

PHLPP

PH domain leucine‐rich repeat protein phosphatase

PP2A

protein phosphatase 2A

qRT‐PCR

quantitative real‐time polymerase chain reaction

SIK2

salt‐inducible kinase 2

TCF/LEF

T cell factor/lymphoid enhancer‐binding factor

TCGA

The Cancer Genome Atlas

     

PRDM14 directly interacts with heat shock proteins HSP90α and glucose‐regulated protein 78

PRDM14 is overexpressed in various cancers and can regulate cancer phenotype under certain conditions. Inhibiting PRDM14 expression in breast and pancreatic cancers has been reported to reduce cancer stem‐like phenotypes, which are associated with aggressive tumor properties. Therefore, PRDM14 is considered a promising target for cancer therapy. To develop a pharmaceutical treatment, the mechanism and interacting partners of PRDM14 need to be clarified. Here, we identified the proteins interacting with PRDM14 in triple‐negative breast cancer (TNBC) cells, which do not express the three most common types of receptor (estrogen receptors, progesterone receptors, and HER2). We obtained 13 candidates that were pulled down with PRDM14 in TNBC HCC1937 cells and identified them by mass spectrometry. Two candidates—glucose‐regulated protein 78 (GRP78) and heat shock protein 90‐α (HSP90α)—were confirmed in immunoprecipitation assay in two TNBC cell lines (HCC1937 and MDA‐MB231). Surface plasmon resonance analysis using GST‐PRDM14 showed that these two proteins directly interacted with PRDM14 and that the interactions required the C‐terminal region of PRDM14, which includes zinc finger motifs. We also confirmed the interactions in living cells by NanoLuc luciferase‐based bioluminescence resonance energy transfer (NanoBRET) assay. Moreover, HSP90 inhibitors (17DMAG and HSP990) significantly decreased breast cancer stem‐like CD24⁻ CD44⁺ and side population (SP) cells in HCC1937 cells, but not in PRDM14 knockdown HCC1937 cells. The combination of the GRP78 inhibitor HA15 and PRDM14 knockdown significantly decreased cell proliferation and SP cell number in HCC1937 cells. These results suggest that HSP90α and GRP78 interact with PRDM14 and participate in cancer regulation.     

Glutamine depletion by crisantaspase hinders the growth of human hepatocellular carcinoma xenografts

Background:

A subset of human hepatocellular carcinomas (HCC) exhibit mutations of β-catenin gene CTNNB1 and overexpress Glutamine synthetase (GS). The CTNNB1-mutated HCC cell line HepG2 is sensitive to glutamine starvation induced in vitro with the antileukemic drug Crisantaspase and the GS inhibitor methionine-L-sulfoximine (MSO).

Methods:

Immunodeficient mice with subcutaneous xenografts of the CTNNB1-mutated HCC cell lines HepG2 and HC-AFW1 were treated with Crisantaspase and/or MSO, and tumour growth was monitored. At the end of treatment, tumour weight and histology were assessed. Serum and tissue amino acids were determined by HPLC. Gene and protein expression were estimated with RT-PCR and western blot and GS activity with a colorimetric method. mTOR activity was evaluated from the phosphorylation of p70S6K1.

Results:

Crisantaspase and MSO depleted serum glutamine, lowered glutamine in liver and tumour tissue, and inhibited liver GS activity. HepG2 tumour growth was significantly reduced by either Crisantaspase or MSO, and completely suppressed by the combined treatment. The combined treatment was also effective against xenografts of the HC-AFW1 cell line, which is Crisantaspase resistant in vitro.

Conclusions:

The combination of Crisantaspase and MSO reduces glutamine supply to CTNNB1-mutated HCC xenografts and hinders their growth.     

PGK1-coupled HSP90 stabilizes GSK3β expression to regulate the stemness of breast cancer stem cells

Objective: Glycogen synthase kinase-3β(GSK3β) has been recognized as a suppressor of Wnt/β-catenin signaling, which is critical for the stemness maintenance of breast cancer stem cells. However, the regulatory mechanisms of GSK3β protein expression remain elusive.Methods: Co-immunoprecipitation and mass spectral assays were performed to identify molecules binding to GSK3β, and to characterize the interactions of GSK3β, heat shock protein 90(Hsp90), and co-chaperones. The role of PGK1 in Hsp90 ch...     

Geldanamycin-mediated inhibition of heat shock protein 90 partially activates dendritic cells,but interferes with their full maturation,accompanied by impaired upregulation of RelB

Background

The chaperon heat shock protein 90 (HSP90) constitutes an important target for anti-tumor therapy due to its essential role in the stabilization of oncogenes. However, HSP90 is ubiquitously active to orchestrate protein turnover, chemotherapeutics that target HSP90 may affect immune cells as a significant side effect. Therefore, we asked for potential effects of pharmacological HSP90 inhibition at a therapeutically relevant concentration on human dendritic cells (DCs) as main inducers of both cellular and humoral immune responses, and on human CD4⁺ T cells as directly activated by DCs and essential to confer B cell help.

Methods

Unstimulated human monocyte-derived DCs (MO-DCs) were treated with the prototypical HSP90 inhibitor geldanamycin (GA). Based on dose titration studies performed to assess cytotoxic effects, GA was applied at a rather low concentration, comparable to serum levels of clinically used HSP90 inhibitors. The immuno-phenotype (surface markers, cytokines), migratory capacity, allo T cell stimulatory and polarizing properties (proliferation, cytokine pattern) of GA-treated MO-DCs were assessed. Moreover, effects of GA on resting and differentially stimulated CD4⁺ T cells in terms of cytotoxicity and proliferation were analysed.

Results

GA induced partial activation of unstimulated MO-DCs. In contrast, when coapplied in the course of MO-DC stimulation, GA prevented the acquisition of a fully mature DC phenotype. Consequently, this MO-DC population exerted lower allo CD4⁺ T cell stimulation and cytokine production. Furthermore, GA exerted no cytotoxic effect on resting T cells, but abrogated proliferation of T cells stimulated by MO-DCs at either state of activation or by stimulatory antibodies.

Conclusion

HSP90 inhibitors at clinically relevant concentrations may modulate adaptive immune responses both on the level of DC activation and T cell proliferation. Surprisingly, unstimulated DCs may be partially activated by that agent. However, due to the potent detrimental effects of HSP90 inhibitors on stimulated CD4⁺ T cells, as an outcome a patients T cell responses might be impaired. Therefore, HSP90 inhibitors most probably are not suitable for treatment in combination with immunotherapeutic approaches aimed to induce DC/T cell activation.     

Morphine promotes angiogenesis by activating PI3K/Akt/HIF-1α pathway and upregulating VEGF in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is characterized by the neo-angiogenesis induced by tumor and adjacent cells. It is a leading cancer-related cause of death. Morphine has effects on angiogenesis with pro-angiogenic or anti-angiogenic phonotypes. This study explores the function of morphine on cancer cell growth, angiogenesis and the underlying mechanism in HCC.MethodsMorphine was used to treat BEL-7402 or HCC-LM3 cells and human umbilical vein endothelial cells (HUVECs) were subsequently incubated in the conditioned media (CM) of HCC cells. The potential effects of cell proliferation, migration and tube formation of CM-treated HUVECs were investigated. Furthermore, the angiogenesis regulated factors of VEGFA, PIGF, ANG-1, ANG-2, FGF-1 and FGF-2 were assessed. siRNA and LY294002 were further used to explore the mechanism mediating the effects of morphine on the angiogenesis pathway. The neovascularization effect by morphine was confirmed through the use of human HCC cancer heterotopic mouse model in vivo.ResultsA significantly increased cell proliferation, migration, and tube formation effect of HUVECs induced by the CM from HCC cell lines treated with morphine was observed. More VEGFA secretion in CM from LM3 or BEL-7402 cell lines was found than the controls (P=0.03 and P=0.027, respectively). VEGFA knock-down could significantly reverse cell proliferation, migration and tube formation induced by the CM from HCC cell lines with morphine treatment. Further molecular experiments indicated that VEGFA secretion was activated by morphine potentially through the PI3K/Akt/HIF-1α pathway. Morphine-induced neovascularization was also observed by the IHC of CD31 and VEGFA.ConclusionsMorphine promotes angiogenesis in hepatocellular carcinoma possibly through the activation of the PI3K/Akt/HIF-1α pathway and VEGFA stimulation.     

miR-548c-5p inhibits proliferation and migration and promotes apoptosis in CD90+ HepG2 cells

Background

Since the introduction of the theory of tumour stem cells (TSCs), the liver cancer stem cell (LCSC)-like cells have become one of the focuses in the research on liver cancer.

Materials and methods.

In this study, CD90⁺ cells were applied as the possible LCSC-like cells, and the miRNA and gene expression were analyzed in the CD90⁺ HepG2 cells. The pilot study showed miR-548c-5p exerted potential effect on the CD90⁺ HepG2 cells and was thereafter applied for the further study. CD90⁺ HepG2 cells were assigned to miR-548c-5p mimic transfection group and control group. MTT assay was performed to detect the proliferation of CD90⁺ HepG2 cells. The migration and invasion abilities were examined by wound healing assay and transwell migration assay, respectively. A detection of apoptosis was performed by fluorescence microscopy.

Results

Our results showed that caspase-3 and bcl-2 were down-regulated while caspase-8 was up-regulated in the CD90⁺ HepG2 cells. Moreover, the miR-548c-5p transfection could down-regulate the expression of β-catenin, Tg737, bcl-2, bcl-XL, and caspase-3, inhibit the proliferation, migration and invasion and promote the apoptosis of the CD90⁺ HepG2 cells.

Conclusions

Our findings indicate the imbalance between apoptosis and anti-apoptosis in the LCSC-like cells, which influence the biological features of LCSC-like cells. miRNA plays a regulatory role in the LCSC-like cells among which miR-548c-5p might be a suppressor.