The rarity of ALDH+ cells is the key to separation of normal versus leukemia stem cells by ALDH activity in AML patients

To understand the precise disease driving mechanisms in acute myeloid leukemia (AML), comparison of patient matched hematopoietic stem cells (HSC) and leukemia stem cells (LSC) is essential. In this analysis, we have examined the value of aldehyde dehydrogenase (ALDH) activity in combination with CD34 expression for the separation of HSC from LSC in 104 patients with de novo AML. The majority of AML patients (80 out of 104) had low percentages of cells with high ALDH activity (ALDH⁺ cells; <1.9%; ALDH‐rare AML), whereas 24 patients had relatively numerous ALDH⁺ cells (≥1.9%; ALDH‐numerous AML). In patients with ALDH‐rare AML, normal HSC could be separated by their CD34⁺ALDH⁺ phenotype, whereas LSC were exclusively detected among CD34⁺ALDH^? cells. For patients with ALDH‐numerous AML, the CD34⁺ALDH⁺ subset consisted mainly of LSC and separation from HSC was not feasible. Functional analyses further showed that ALDH⁺ cells from ALDH‐numerous AML were quiescent, refractory to ARA‐C treatment and capable of leukemic engraftment in a xenogenic mouse transplantation model. Clinically, resistance to chemotherapy and poor long‐term outcome were also characteristic for patients with ALDH‐numerous AML providing an additional risk‐stratification tool. The difference in spectrum and relevance of ALDH activity in the putative LSC populations demonstrates, in addition to phenotypic and genetic, also functional heterogeneity of leukemic cells and suggests divergent roles for ALDH activity in normal HSC versus LSC. By acknowledging these differences our study provides a new and useful tool for prospective identification of AML cases in which separation of HSC from LSC is possible.     

Disulfiram modulates stemness and metabolism of brain tumor initiating cells in atypical teratoid/rhabdoid tumors

Background

Atypical teratoid/rhabdoid tumors (AT/RT) are among the most malignant pediatric brain tumors. Cells from brain tumors with high aldehyde dehydrogenase (ALDH) activity have a number of characteristics that are similar to brain tumor initiating cells (BTICs). This study aimed to evaluate the therapeutic potential of ALDH inhibition using disulfiram (DSF) against BTICs from AT/RT.

Methods

Primary cultured BTICs from AT/RT were stained with Aldefluor and isolated by fluorescence activated cell sorting. The therapeutic effect of DSF against BTICs from AT/RT was confirmed in vitro and in vivo.

Results

AT/RT cells displayed a high expression of ALDH. DSF demonstrated a more potent cytotoxic effect on ALDH⁺ AT/RT cells compared with standard anticancer agents. Notably, treatment with DSF did not have a considerable effect on normal neural stem cells or fibroblasts. DSF significantly inhibited the ALDH enzyme activity of AT/RT cells. DSF decreased self-renewal ability, cell viability, and proliferation potential and induced apoptosis and cell cycle arrest in ALDH⁺ AT/RT cells. Importantly, DSF reduced the metabolism of ALDH⁺ AT/RT cells by increasing the nicotinamide adenine dinucleotide ratio of NAD⁺/NADH and regulating Silent mating type Information Regulator 2 homolog 1 (SIRT1), nuclear factor-kappaB, Lin28A/B, and miRNA let-7g. Animals in the DSF-treated group demonstrated a reduction of tumor volume (P < .05) and a significant survival benefit (P = .02).

Conclusion

Our study demonstrated the therapeutic potential of DSF against BTICs from AT/RT and suggested the possibility of ALDH inhibition for clinical application.     

ALDH/CD44 identifies uniquely tumorigenic cancer stem cells in salivary gland mucoepidermoid carcinomas

A small sub-population of cells characterized by increased tumorigenic potential, ability to self-renew and to differentiate into cells that make up the tumor bulk, has been characterized in some (but not all) tumor types. These unique cells, namedcancer stem cells, are considered drivers of tumor progression in these tumors. The purpose of this work is to understand if cancer stem cells play a functional role in the tumorigenesis of salivary gland mucoepidermoid carcinomas. Here, we investigated the expression of putative cancer stem cell markers (ALDH, CD10, CD24, CD44) in primary human mucoepidermoid carcinomas by immunofluorescence, in vitro salisphere assays, and in vivo tumorigenicity assays in immunodeficient mice. Human mucoepidermoid carcinoma cells (UM-HMC-1, UM-HMC-3A, UM-HMC-3B) sorted for high levels of ALDH activity and CD44 expression (ALDH^highCD44^high) consistently formed primary and secondary salispheres in vitro, and showed enhanced tumorigenic potential in vivo (defined as time to tumor palpability, tumor growth after palpability), when compared to ALDH^lowCD44^low cells. Cells sorted for CD10/CD24, and CD10/CD44 showed varying trends of salisphere formation, but consistently low in vivo tumorigenic potential. And finally, cells sorted for CD44/CD24 showed inconsistent results in salisphere formation and tumorigenic potential assays when different cell lines were evaluated. Collectively, these data demonstrate that salivary gland mucoepidermoid carcinomas contain a small population of cancer stem cells with enhanced tumorigenic potential and that are characterized by high ALDH activity and CD44 expression. These results suggest that patients with mucoepidermoid carcinoma might benefit from therapies that ablate these highly tumorigenic cells.     

Malignant phyllodes tumors display mesenchymal stem cell features and aldehyde dehydrogenase/disialoganglioside identify their tumor stem cells

Introduction

Although breast phyllodes tumors are rare, there is no effective therapy other than surgery. Little is known about their tumor biology. A malignant phyllodes tumor contains heterologous stromal elements, and can transform into rhabdomyosarcoma, liposarcoma and osteosarcoma. These versatile properties prompted us to explore their possible relationship to mesenchymal stem cells (MSCs) and to search for the presence of cancer stem cells (CSCs) in phyllodes tumors.

Methods

Paraffin sections of malignant phyllodes tumors were examined for various markers by immunohistochemical staining. Xenografts of human primary phyllodes tumors were established by injecting freshly isolated tumor cells into the mammary fat pad of non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice. To search for CSCs, xenografted tumor cells were sorted into various subpopulations by flow cytometry and examined for their in vitro mammosphere forming capacity, in vivo tumorigenicity in NOD-SCID mice and their ability to undergo differentiation.

Results

Immunohistochemical analysis revealed the expression of the following 10 markers: CD44, CD29, CD106, CD166, CD105, CD90, disialoganglioside (GD2), CD117, Aldehyde dehydrogenase 1 (ALDH), and Oct-4, and 7 clinically relevant markers (CD10, CD34, p53, p63, Ki-67, Bcl-2, vimentin, and Globo H) in all 51 malignant phyllodes tumors examined, albeit to different extents. Four xenografts were successfully established from human primary phyllodes tumors. In vitro, ALDH⁺ cells sorted from xenografts displayed approximately 10-fold greater mammosphere-forming capacity than ALDH^- cells. GD2⁺ cells showed a 3.9-fold greater capacity than GD2^- cells. ALDH⁺/GD2⁺cells displayed 12.8-fold greater mammosphere forming ability than ALDH^-/GD2^- cells. In vivo, the tumor-initiating frequency of ALDH⁺/GD2⁺ cells were up to 33-fold higher than that of ALDH⁺ cells, with as few as 50 ALDH⁺/GD2⁺ cells being sufficient for engraftment. Moreover, we provided the first evidence for the induction of ALDH⁺/GD2⁺ cells to differentiate into neural cells of various lineages, along with the observation of neural differentiation in clinical specimens and xenografts of malignant phyllodes tumors. ALDH⁺ or ALDH⁺/GD2⁺ cells could also be induced to differentiate into adipocytes, osteocytes or chondrocytes.

Conclusions

Our findings revealed that malignant phyllodes tumors possessed many characteristics of MSC, and their CSCs were enriched in ALDH⁺ and ALDH⁺/GD2⁺ subpopulations.     

Cancer stem cell related markers of radioresistance in head and neck squamous cell carcinoma

Despite recent advances in understanding of the molecular pathogenesis and improvement of treatment techniques, locally advanced head and neck squamous cell carcinoma (HNSCC) remains associated with an unfavorable prognosis. Compelling evidence suggests that cancer stem cells (CSC) may cause tumor recurrence if they are not eradicated by current therapies as radiotherapy or radio-chemotherapy. Recent in vitro studies have demonstrated that CSCs may be protected from treatment-induced death by multiple intrinsic and extrinsic mechanisms. Therefore, early determination of CSC abundance in tumor biopsies prior-treatment and development of therapeutics, which specifically target CSCs, are promising strategies to optimize treatment. Here we provide evidence that aldehyde dehydrogenase (ALDH) activity is indicative for radioresistant HNSCC CSCs. Our study suggests that ALDH⁺ cells comprise a population that maintains its tumorigenic properties in vivo after irradiation and may provide tumor regrowth after therapy. We found that ALDH activity in HNSCC cells can be attributed, at least in part, to the ALDH1A3 isoform and inhibition of the ALDH1A3 expression by small interfering RNA (siRNA) decreases tumor cell radioresistance. The expression dynamic of ALDH1A3 upon irradiation by either induction or selection of the ALDH1A3 positive population correlates to in vivo curability, suggesting that changes in protein expression during radiotherapy are indicative for tumor radioresistance. Our data indicate that ALDH1A3⁺ HNSCC cells may contribute to tumor relapse after irradiation, and inhibition of this cell population might improve therapeutic response to radiotherapy.     

Prospective identification of tumorigenic osteosarcoma cancer stem cells in OS99‐1 cells based on high aldehyde dehydrogenase activity

High aldehyde dehydrogenase (ALDH) activity has recently been used to identify tumorigenic cell fractions in many cancer types. Herein we hypothesized that a subpopulation of cells with cancer stem cells (CSCs) properties could be identified in established human osteosarcoma cell lines based on high ALDH activity. We previously showed that a subpopulation of cells with high ALDH activity were present in 4 selected human osteosarcoma cell lines, of which a significantly higher ALDH activity was present in the OS99‐1 cell line that was originally derived from a highly aggressive primary human osteosarcoma. Using a xenograft model in which OS99‐1 cells were grown in NOD/SCID mice, we identified a highly tumorigenic subpopulation of osteosarcoma cells based on their high ALDH activity. Cells with high ALDH activity (ALDH^br cells) from the OS99‐1 xenografts were much less frequent, averaging 3% of the entire tumor population, compared to those isolated directly from the OS99‐1 cell line. ALDH^br cells from the xenograft were enriched with greater tumorigenicity compared to their counterparts with low ALDH activity (ALDH^lo cells), generating new tumors with as few as 100 cells in vivo. The highly tumorigenic ALDH^br cells illustrated the stem cell characteristics of self‐renewal, the ability to produce differentiated progeny and increased expression of stem cell marker genes OCT3/4A, Nanog and Sox‐2. The isolation of osteosarcoma CSCs by their high ALDH activity may provide new insight into the study of osteosarcoma‐initiating cells and may potentially have therapeutic implications for human osteosarcoma.     

Tumor endothelial cells with high aldehyde dehydrogenase activity show drug resistance

Tumor blood vessels play an important role in tumor progression and metastasis. We previously reported that tumor endothelial cells (TEC) exhibit several altered phenotypes compared with normal endothelial cells (NEC). For example, TEC have chromosomal abnormalities and are resistant to several anticancer drugs. Furthermore, TEC contain stem cell‐like populations with high aldehyde dehydrogenase (ALDH) activity (ALDH^high TEC). ALDH^high TEC have proangiogenic properties compared with ALDH^low TEC. However, the association between ALDH^high TEC and drug resistance remains unclear. In the present study, we found that ALDH mRNA expression and activity were higher in both human and mouse TEC than in NEC. Human NEC:human microvascular endothelial cells (HMVEC) were treated with tumor‐conditioned medium (tumor CM). The ALDH^high population increased along with upregulation of stem‐related genes such as multidrug resistance 1, CD90, ALP, and Oct‐4. Tumor CM also induced sphere‐forming ability in HMVEC. Platelet‐derived growth factor (PDGF)‐A in tumor CM was shown to induce ALDH expression in HMVEC. Finally, ALDH^high TEC were resistant to fluorouracil (5‐FU) in vitro and in vivo. ALDH^high TEC showed a higher grade of aneuploidy compared with that in ALDH^low TEC. These results suggested that tumor‐secreting factor increases ALDH^high TEC populations that are resistant to 5‐FU. Therefore, ALDH^high TEC in tumor blood vessels might be an important target to overcome or prevent drug resistance.     

Magnetic resonance spectroscopy suggests key differences in the metastatic behaviour of medulloblastoma

BackgroundMetastatic medulloblastoma has a poorer prognosis than localised disease in part due to inherent properties of the tumour. ¹H magnetic resonance spectroscopy (MRS) provides a powerful method for investigating tumour metabolism in vivo.MethodsMagnetic resonance imaging and short echo time (Te 30 ms) single voxel MRS were performed on the primary tumour of 16 children with medulloblastoma prior to surgical resection. Tumour volumes were calculated using a segmentation technique and the MRS was analysed using LCModel™.ResultsPatients with metastatic disease had primary tumours which were smaller (p = 0.01), had higher levels of total choline (p = 0.03) and lower levels of mobile lipids (p = 0.04).ConclusionMetastatic medulloblastomas have metabolite profiles indicative of increased cell growth and decreased cell death compared with localised tumours reflecting intrinsic differences in underlying biology. Localised tumours with an MRS metabolite profile similar to those with metastatic disease may be at increased risk of metastatic relapse.     

Magnetic resonance spectroscopy in the assessment of pilocytic astrocytomas

BackgroundPilocytic astrocytomas (PA) are common childhood brain tumours whose management and prognosis vary widely depending on location. ¹H magnetic resonance spectroscopy (MRS) measures biochemistry in vivo and shows promise for characterising brain tumours and aiding management.MethodsSingle voxel MRS (1.5 Tesla, TE 30 ms, TR 1500 ms) was performed on 27 children with PAs. Cases were designated ‘progressors’ if tumour progression led to their clinical management plan being altered.ResultsPrior to treatment, supratentorial tumours had significantly higher myo-inositol (p < 0.01, t-test) and glutamate plus glutamine (p = 0.02, t-test) than cerebellar tumours. Optic pathway or thalamic tumours that progressed had a significantly (p = 0.04, t-test) lower myo-inositol at initial MRS than those with stable disease. Myo-inositol levels decreased significantly in progressors between the initial and subsequent MRS (p = 0.03, paired t-test). Changes in myo-inositol occurred before clinical and radiological progression.ConclusionsMRS identifies differences with anatomical location in PAs and yields potential non-invasive biomarkers of prognosis.     

HER3 expression in patients with primary colorectal cancer and corresponding lymph node metastases related to clinical outcome

AimTo evaluate the expression and prognostic value of the epidermal growth factor receptor HER3 in patients with primary colorectal cancer (CRC) and corresponding lymph node metastases.Patient and methodsHER3 expression was analysed immunohistochemically (IHC) in primary tumours and in corresponding lymph node metastases from 236 patients with stage II and III CRC. In 58 primary tumours, fluorescence in situ hybridisation (FISH) detection was performed.ResultsHER3 was detected at high frequency in the cell membrane. Seventy percent of the primary tumours had a high HER3 expression compared to 75% in the lymph node metastases. HER3 expression in the primary tumour was an independent prognostic factor for overall survival in the entire group of patients (p = 0.026) and in the subgroup of patients with colon cancer stage II (p = 0.030). A high HER3 expression in the primary tumour was associated with worse clinical outcome. The expression of HER3 was homogenous within the primary tumour (r = 0.9, p < 0.0001) and correlated with the HER3 expression in corresponding lymph node metastases (r = 0.6, p < 0.0001). No gene amplification with respect to HER3 was seen in primary tumours using FISH analysis.ConclusionA high HER3 expression was found in 70% of the primary CRC tumours and in 75% of the corresponding lymph node metastases. HER3 expression in the tumour was an independent prognostic factor, where a high HER3 expression was associated with worse clinical outcome. There was a correlation in HER3 expression between primary tumour and corresponding lymph node metastases.     

Tumour CD133 mRNA expression and clinical outcome in surgically resected colorectal cancer patients

BackgroundHuman prominin-1 (CD133) is a novel pentaspan membrane protein which was originally classified as a marker of primitive haematopoietic and neural stem cells. Cancer stem cells have been isolated and expanded from leukaemia and several solid tumours, and have been associated with metastasis, chemoresistance and relapse. CD133 is recognised as a stem cell marker and is capable of identifying a tumour-initiating subpopulation in brain, colon, melanoma and other solid tumours.MethodsWe assessed CD133 mRNA expression levels by RT-QPCR in tumour and matched normal tissue from 64 stages I–III colorectal cancer (CRC) patients and correlated tumour CD133 levels with clinicopathological characteristics and clinical outcome.ResultsIn four patients, CD133 mRNA was not expressed in tumour or in normal tissue. In the remaining 60 patients, expression levels were higher in tumour than in normal tissue (p = 0.001). Higher levels of CD133 expression were associated with shorter relapse-free interval (RFI) (p = 0.004) and overall survival (OS) (p < 0.0001). In the multivariate analyses, CD133 levels emerged as a prognostic marker for RFI and OS.ConclusionsWe have observed longer RFI and OS in patients with lower levels of CD133, regardless of adjuvant treatment and other clinical characteristics. If these findings are confirmed in larger prospective studies, CD133 assessment may prove useful for new diagnostic and therapeutic procedures for CRC patients.     

Cancer stem cell enrichment marker CD98: A prognostic factor for survival in patients with human papillomavirus-positive oropharyngeal cancer

PurposeSeveral hypotheses have been proposed to explain the relatively good prognosis of patients with a human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) and one of these is a higher sensitivity to (chemo)radiation. Previous studies have suggested that treatment failure in OPSCC patients is caused by resistance of cancer stem cells (CSCs). The purpose of this study was to evaluate the association between the number of CSCs and prognosis in HPV-positive OPSCC patients.Experimental designAll OPSCC patients (n = 711) treated between 2000 and 2006 in two Dutch university hospitals were included. Presence of HPV in a tumour tissue specimen was tested by p16-immunostaining followed by HPV DNA GP5+/6+polymerase chain reaction (PCR). The presence and intensity of tumour CSC markers CD44 and CD98 were determined by immunohistochemistry and semiquantitative scoring was performed. Overall survival (OS) and progression-free survival (PFS) rates were compared between patients with low and high CD44/CD98 expression in relation to HPV status.ResultsHPV-positive tumours showed a lower percentage of cells with CD44 and CD98 expression than HPV-negative tumours (p < 0.001, χ²-test). Within the group of patients with HPV-positive OPSCC, a high percentage of CD98-positive tumour cells was associated with a significantly worse 5-year OS and PFS (OS: 36.4% and PFS: 27.3%) compared to patients with a low percentage of CD98-positive cells (OS: 71.9% and PFS: 70.5%, respectively) (p < 0.001).ConclusionsHPV-positive OPSCCs harbour fewer cells expressing the CSC enrichment markers CD44 and CD98. Furthermore, OS and PFS were significantly worse for patients with HPV-positive OPSCC with a high percentage of CD98-positive cells.     

Programmed death-1 ligand 1 and 2 are highly expressed in pleomorphic carcinomas of the lung: Comparison of sarcomatous and carcinomatous areas

Pleomorphic carcinoma (PC) of the lung is a rare type of poorly differentiated non-small cell lung carcinoma (NSCLC) that belongs to sarcomatoid carcinoma (SC). It exhibits aggressive behaviour and resistance to chemotherapy and radiotherapy. Recently, immunotherapy targeting the programmed death-1 (PD-1)/PD ligand 1 (PD-L1) pathway has demonstrated favourable clinical outcomes in NSCLC. However, the expression patterns of PD-1-related molecules in pulmonary PC remain elusive.PD-L1 and PD-L2 expression was estimated in 41 cases of PC using immunohistochemistry. CD8⁺ and PD-1⁺ tumour-infiltrating lymphocytes (TILs) were also evaluated.PD-L1 and PD-L2 were highly expressed in pulmonary PCs (90.2% [37/41)]; 87.8% [36/41]). The amount of CD8⁺ or PD-1⁺ TILs and the ratio of PD-1⁺/CD8⁺ TILs in PC were higher in males, smokers and older patients. PD-L1-positive PCs were infiltrated by higher numbers of CD8⁺ TILs compared to PD-L1-negative cases (P = 0.006). Of note, PD-L1 expression in pulmonary PCs was significantly higher in sarcomatous areas than in the carcinomatous portion (P = 0.006). PC patients with a high ratio of PD-1⁺/CD8⁺ TILs showed a shorter progression-free survival (P = 0.036), whereas PD-L1 and PD-L2 expression had no prognostic implications.Our study demonstrates that pulmonary PCs very frequently express PD-L1 and PD-L2. Moreover, their expression is higher in sarcomatous cells than in carcinomatous areas. Thus, targeting the PD-1/PD-L1 pathway may represent a potential therapeutic candidate for this aggressive tumour.     

Anti-insulin-like growth factor 1 receptor antibody EM164 (murine AVE1642) exhibits anti-tumour activity alone and in combination with temozolomide against neuroblastoma

Insulin-like growth factor 1 receptor (IGF-1R) is overexpressed in many tumours and contributes to tumourigenicity, cell proliferation, metastasis and resistance, thus representing a promising therapeutic target. The human IGF-1R antagonistic monoclonal antibody EM164 (murine AVE1642) has shown activity in adult cancers and is being evaluated in patients with advanced malignancies. We investigated the EM164 for its therapeutic potential against childhood neuroblastoma.EM164 at 0.07, 0.7 and 7 μg/mL exhibited anti-proliferative activity against all nine cell lines tested in ³H-thymidine incorporation assay in vitro. Cell proliferation after EM164 exposure ranged between 24% and 80% compared to controls. Sensitivity was independent from culture serum conditions, intensity of IGF-1R expression and IGF-II secretion, although associated with inhibition of AKT activation. In vivo, EM164 administered intravenously at 40 mg/kg twice weekly for 4 weeks yielded significant tumour growth delays (TGD) of 13.4 d in advanced stage IGR-N91 and 12.9 d in SK-N-AS tumours compared to controls (p = 0.02 and p = 0.0059, respectively). Simultaneous treatment of EM164 0.7 μg/mL and temozolomide resulted in enhanced activity in vitro. In vivo, treatment with temozolomide at the maximum tolerated dose (100 mg/kg/d for 5 consecutive days) and EM164 yielded a significantly greater TGD of 29.1 d (p < 0.01) and two complete tumour regressions (CR) compared to 18.1 d (p = ns) and one CR for EM164 alone and 16.1 d (p = ns) for temozolomide alone.Our results demonstrate the potential of the anti-IGF-1R antibody alone and in combination with alkylating agents and support the therapeutic development of the AVE1642 for aggressive neuroblastoma.     

Enhancement of tumor initiation and expression of KCNMA1, MORF4L2 and ASPM genes in the adenocarcinoma of lung xenograft after vorinostat treatment

Cancer stem cells (CSCs) are usually tolerant to chemotherapy and radiotherapy and associated with tumor relapse. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor (HDACI), is currently being used in clinical trials of lung cancer. However, SAHA facilitates the formation of induced pluripotent stem cells from somatic cells. We hypothesized that SAHA would mediate the CSCs properties and subsequently confer a more malignant phenotype in lung cancer. Transfected H1299 lung cancer cells, which stably expresses a triple fused reporter gene (DsRedm-Fluc-tTKsr39) under the control of CMV promoter was used to establish a xenograft mouse model. After the treatment of SAHA, H1299 cell line and tumor xenografts were sorted by fluorescence-activated cell sorting (FACS) based on aldehyde dehydrogenase (ALDH) activity. We found that SAHA could suppress the growth of xenografted H1299 tumors with decreased proportion of ALDH^br lung cancer cells indicating that SAHA may target CSCs. However, SAHA significantly enhanced the tumor initiating capacity and the expression of malignant genes such as KCNMA1, MORF4L2 and ASPM in the remaining living ALDH^br cells. These findings suggested that SAHA treatment created a more drug-resistant state in residual ALDH^br cells. The in vivo imaging technique may facilitate searching and characterization of CSCs.     

Gene expression profiling of circulating CD133+ cells of hepatocellular carcinoma patients associated with HCV infection

Aim

Identifying the genetic expression profile of CD133⁺ cells from HCC patients compared to CD133⁺ cells from healthy volunteers that may contribute in hepatocarcinogenesis process.

A STAT3-NFkB/DDIT3/CEBPβ axis modulates ALDH1A3 expression in chemoresistant cell subpopulations

Here we studied the relevance and modulation of aldehyde dehydrogenase (ALDH) expression in malignant pleural mesothelioma (MPM) chemoresistant cell subpopulations (ALDH^bright cells), which survive pemetrexed + cisplatin treatment in vitro and in vivo. Expression of the ALDH1A3 isoform was invariably enriched in purified ALDH^bright cells from multiple MPM cell lines and accounted for the enzymatic activity of those cells. RNAi mediated downregulation of ALDH1A3 reduced the survival of the ALDH^bright cells at steady state and, much more, after pemetrexed + cisplatin treatment. We demonstrated, for the first time, that a pSTAT3(tyr705)-NFkB(p65) complex is required for the repression of DDIT3 mRNA and this ensures high levels of CEBPβ-dependent ALDH1A3 promoter activity. Inhibition of STAT3-NFkB activity allowed high levels of DDIT3 expression with increased formation of a DDIT3-CEBPβ complex. This reduced the occupancy of the ALDH1A3 promoter by CEBPβ, thus largely reducing the ALDH1A3 expression. Consequently, survival of ALDH^bright cells in pemetrexed + cisplatin-treated cultures was impaired, following increased apoptosis. We show that such a mechanism is relevant in vivo and underlies the action of butein, a dual STAT3-NFkB inhibitor capable of abating the chemoresistance of mesothelioma cells in vivo. The possible broad translational relevance of the described mechanism is discussed.     

Adenoviral vector expressing short hairpin RNA targeting Wnt2B has an effective antitumour activity against Wnt2B2-overexpressing tumours

BackgroundThe Wnt family encodes multi-functional signalling glycoproteins regulating various normal and pathological processes including tumourigenesis. Wnt2B overexpression is thought to affect tumour progression through the activation of the canonical Wnt pathway.MethodExperimental studies were conducted using a Wnt2B-inhibiting vector to establish gene therapy against Wnt2B2-overexpressing tumours. A replication-deficient recombinant adenoviral vector expressing short hairpin RNA targeting Wnt2B (Ad-shWnt2B) was constructed. Three Wnt2B2-overexpressing human tumour cells, including A549 cells, Hela cells and PANC1 cells, were used. Thereafter, cell viability was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Next, a human tumour xenograft model in nude mice was prepared by subcutaneously implanting tumours derived from A549 cells. Ad-shWnt2B was administered via intratumoural injection every 4 days.ResultsFirst, immunohistochemical studies revealed that high levels of Wnt2B expression appeared in proliferative normal tissues and many human tumour tissues. Furthermore, the Wnt2B2 gene expression was associated with c-Myc and survivin expressions in human lung cancer. Transduction with Ad-shWnt2B effectively downregulated the Wnt2B2 expression in all the three Wnt2B2-overexpressing tumour cells (p < 0.0001). The transduction with Ad-shWnt2B significantly reduced the percentage of viable cells in all the Wnt2B2-overexpressing tumour cells (p < 0.005). In addition, transduction with Ad-shWnt2B significantly downregulated c-Myc and survivin in A549 cells (p < 0.005). Furthermore, the treatment with Ad-shWnt2B exerted a significant antitumour effect against the Wnt2B2-overexpressing A549 xenografts by inducing apoptosis (p < 0.01).ConclusionsCancer gene therapy using an adenoviral vector expressing short hairpin RNA (shRNA) against Wnt2B was, therefore, found to have a strong antitumour effect against Wnt2B2-overexpressing tumours.