Modulation of tumorigenesis and oestrogen receptor‐α expression by cell culture conditions in a stem cell‐derived breast epithelial cell line

Background information. The common phenotypes of cancer and stem cells suggest that cancers arise from stem cells. Oestrogen is one of the few most important determinants of breast cancer, as shown by several lines of convincing evidence. We have previously reported a human breast epithelial cell type (Type 1 HBEC) with stem cell characteristics and ERα (oestrogen receptor α) expression. A tumorigenic cell line, M13SV1R2, was developed from this cell type after SV40 (simian virus 40) large T‐antigen transfection and X‐ray irradiation. The cell line, however, was not responsive to oestrogen for cell growth or tumour development. In the present study, we tested the hypothesis that deprivation of growth factors and hormones may change the tumorigenicity and oestrogen response of this cell line. Results. The M13SV1R2 cells lost their tumorigenicity after culturing in a growth factor/hormone‐deprived medium for >10 passages (referred to as R2d cells) concomitant with the expression of two tumour suppressor genes, namely those coding for maspin and α6 integrin. However, these cells acquired oestrogen responsiveness in cell growth and tumour development. By immunocytochemistry, Western blotting and flow cytometry analysis, oestrogen treatment of R2d cells was found to induce many important effects related to breast carcinogenesis, namely: (i) the emergence of a subpopulation of cells expressing CD44^+/high/CD24^?/low breast tumour stem cell markers; (ii) the induction of EMT (epithelial‐to‐mesenchymal transition); (iii) the acquisition of metastatic ability; and (iv) the expression of COX‐2 (cyclo‐oxygenase‐2) through a CD44‐mediated mechanism. Conclusion. An oestrogen‐responsive cell line with ERα and CD44⁺/CD24^?/low expression can be derived from breast epithelial stem cells. The tumorigenicity and oestrogen response of these cells could depend on the cell culture conditions. The findings of this study have implications in regard to the origins of (1) ERα‐positive breast cancers, (2) CD44⁺/CD24^?/low breast tumour stem cells and (3) the metastatic ability of breast cancer.     

Silencing of Prrx1b suppresses cellular proliferation,migration, invasion and epithelial–mesenchymal transition in triple‐negative breast cancer

Triple‐negative breast cancer (TNBC) is a highly aggressive tumour subtype associated with poor prognosis. The mechanisms involved in TNBC progression remains largely unknown. To date, there are no effective therapeutic targets for this tumour subtype. Paired‐related homeobox 1b (Prrx1b), one of major isoforms of Prrx1, has been identified as a new epithelial–mesenchymal transition (EMT) inducer. However, the function of Prrx1b in TNBC has not been elucidated. In this study, we found that Prrx1b was significantly up‐regulated in TNBC and associated with tumour size and vascular invasion of breast cancer. Silencing of Prrx1b suppressed the proliferation, migration and invasion of basal‐like cancer cells. Moreover, silencing of Prrx1b prevented Wnt/β‐catenin signaling pathway and induced the mesenchymal‐epithelial transition (MET). Taken together, our data indicated that Prrx1b may be an important regulator of EMT in TNBC cells and a new therapeutic target for interventions against TNBC invasion and metastasis.     

Molecular characterization of circulating tumour cells identifies predictive markers for outcome in primary,triple‐negative breast cancer patients

mRNA profiles of circulating tumour cells (CTCs) were analysed in patients with triple‐negative breast cancer (TNBC) (pts) before (BT) and after therapy (AT) to identify additional treatment options. 2 × 5 mL blood of 51 TNBC pts and 24 non‐TNBC pts (HR+/HER2?; HR?/HER2+) was analysed for CTCs using the AdnaTest EMT‐2/Stem Cell Select?, followed by mRNA isolation and cDNA analysis for 17 genes by qPCR PIK3CA, AKT2, MTOR and the resistance marker AURKA and ERCC1 were predominantly expressed in all breast cancer subtypes, the latter ones especially AT. In TNBC pts, ERBB3, EGFR, SRC, NOTCH, ALK and AR were uniquely present and ERBB2+/ERBB3 + CTCs were found BT and AT in about 20% of cases. EGFR+/ERBB2+/ERBB3 + CTCs BT and ERBB2+/ERBB3 + CTCs AT significantly correlated with a shorter progression‐free survival (PFS; P = 0.01 and P = 0.02). Platinum‐based therapy resulted in a reduced PFS (P = 0.02) and an induction of PIK3CA expression in CTCs AT. In non‐TNBC pts, BT, the expression pattern in CTCs was similar. AURKA+/ERCC1 + CTCs were found in 40% of HR?/HER2 + pts BT and AT. In the latter group, NOTCH, PARP1 and SRC1 were only present AT and ERBB2 + CTCs completely disappeared AT. These findings might help to predict personalized therapy for TNBC pts in the future.     

Long non‐coding RNA FEZF1‐AS1 promotes breast cancer stemness and tumorigenesis via targeting miR‐30a/Nanog axis

Long non‐coding RNAs (lncRNAs) have been verified to modulate the tumorigenesis of breast cancer at multiple levels. In present study, we aim to investigate the role of lncRNA FEZF1‐AS1 on breast cancer‐stem like cells (BCSC) and the potential regulatory mechanism. In breast cancer tissue, lncRNA FEZF1‐AS1 was up‐regulated compared with controls and indicated poor prognosis of breast cancer patients. In vitro experiments, FEZF1‐AS1 was significantly over‐expressed in breast cancer cells, especially in sphere subpopulation compared with parental subpopulation. Loss‐of‐functional indicated that, in BCSC cells (MDA‐MB‐231 CSC, MCF‐7 CSC), FEZF1‐AS1 knockdown reduced the CD44⁺/CD24^? rate, the mammosphere‐forming ability, stem factors (Nanog, Oct4, SOX2), and inhibited the proliferation, migration and invasion. In vivo, FEZF1‐AS1 knockdown inhibited the breast cancer cells growth. Bioinformatics analysis tools and series of validation experiments confirmed that FEZF1‐AS1 modulated BCSC and Nanog expression through sponging miR‐30a, suggesting the regulation of FEZF1‐AS1/miR‐30a/Nanog. In summary, our study validate the important role of FEZF1‐AS1/miR‐30a/Nanog in breast cancer stemness and tumorigenesis, providing a novel insight and treatment strategy for breast cancer.     

Metformin mediates induction of miR‐708 to inhibit self‐renewal and chemoresistance of breast cancer stem cells through targeting CD47

Breast cancer stem cells (BCSCs) have been considered responsible for cancer progression, recurrence, metastasis and drug resistance. However, the mechanisms by which cells acquire self‐renewal and chemoresistance properties are remaining largely unclear. Herein, we evaluated the role of miR‐708 and metformin in BCSCs, and found that the expression of miR‐708 is significantly down‐regulated in BCSCs and tumour tissues, and correlates with chemotherapy response and prognosis. Moreover, miR‐708 markedly inhibits sphere formation, CD44⁺/CD24^? ratio, and tumour initiation and increases chemosensitivity of BCSCs. Mechanistically, miR‐708 directly binds to cluster of differentiation 47 (CD47), and regulates tumour‐associated macrophage‐mediated phagocytosis. On the other hand, CD47 is essential for self‐renewal, tumour initiation and chemoresistance of BCSCs, and correlates with the prognosis of breast cancer patients. In addition, the anti‐type II diabetes drug metformin are found to be involved in the miR‐708/CD47 signalling pathway. Therefore, our study demonstrated that miR‐708 plays an important tumour suppressor role in BCSCs self‐renewal and chemoresistance, and the miR‐708/CD47 regulatory axis may represent a novel therapeutic mechanism of metformin in BCSCs.     

Progress in the research of p53 tumour suppressor activity controlled by Numb in triple‐negative breast cancer

Numb is known as a cell fate determinant as it identifies the direction of cell differentiation via asymmetrical partitioning during mitosis. It is considered as a tumour suppressor, and a frequent loss of Numb expression in breast cancer is noted. Numb forms a tri‐complex with p53 and E3 ubiquitin ligase HDM2 (also known as MDM2), thereby preventing the ubiquitination and degradation of p53. In this study, we examined Numb expression in 125 patients with triple‐negative breast cancer (TNBC). The results showed that 61 (48.8%) patients presented with a deficient or decreased Numb expression. The percentage of Ki67 > 14% in the retained Numb group was significantly lower than that in the decreased and deficient Numb groups (86.00% vs. 98.40%, P = .0171). This study aimed to detect the expression and migration of Numb, HDM2 and p53 in the membrane, cytoplasmic and nuclear fractions of normal mammary epithelial cell line MCF‐10A and basal‐like TNBC cell line MDA‐MB‐231. We obtained the cell fractions to identify changes in these three protein levels after the re‐expression of NUMB in the MDA‐MB‐231 cells and the knocking down of NUMB in the MCF‐10A cells. Results showed that Numb regulates p53 levels in the nucleus where the protein levels of Numb are positively correlated with p53 levels, regardless if it is re‐expressed in the MDA‐MB‐231 cells or knocked down in the MCF‐10A cells. Moreover, HDM2 was remarkably decreased only in the membrane fraction of NUMB knock‐down cells; however, its mRNA levels were increased significantly. Our results reveal a previously unknown molecular mechanism that Numb can migrate into the nucleus and interact with HDM2 and p53.     

Tumour‐associated macrophages correlate with microvascular bed extension in colorectal cancer patients

Tumour‐associated macrophages (TAMs) represent pivotal components of tumour microenvironment promoting angiogenesis, tumour progression and invasion. In colorectal cancer (CRC), there are no conclusive data about the role of TAMs in angiogenesis‐mediated tumour progression. In this study, we aimed to evaluate a correlation between TAMs, TAM immunostained area (TAMIA) microvascular density (MVD), endothelial area (EA) and cancer cells positive to VEGF‐A (CCP‐VEGF‐A) in primary tumour tissue of locally advanced CRC patients undergone to radical surgery. A series of 76 patients with CRC were selected and evaluated by immunohistochemistry and image analysis. An anti‐CD68 antibody was employed to assess TAMs and TAMIA expression, an anti‐CD34 antibody was utilized to detect MVD and EA expression, whereas an anti‐VEGF‐A antibody was used to detect CCP‐VEGF‐A; then, tumour sections were evaluated by image analysis methods. The mean ± S.D. of TAMs, MVD and CCP‐VEGF‐A was 65.58 ± 21.14, 28.53 ± 7.75 and 63% ± 37%, respectively; the mean ± S.D. of TAMIA and EA was 438.37 ± 124.14μ² and 186.73 ± 67.22μ², respectively. A significant correlation was found between TAMs, TAMIA, MVD and EA each other (r ranging from 0.69 to 0.84; P ranging from 0.000 to 0.004). The high level of expression of TAMs and TAMIA in tumour tissue and the significant correlation with both MVD and EA illustrate that TAMs could represent a marker that plays an important role in promoting angiogenesis‐mediated CRC. In this context, novel agents killing TAMs might be evaluated in clinical trials as a new anti‐angiogenic approach.     

Inhibition of yes‐associated protein suppresses brain metastasis of human lung adenocarcinoma in a murine model

Yes‐associated protein (YAP) is a main mediator of the Hippo pathway and promotes cancer development and progression in human lung cancer. We sought to determine whether inhibition of YAP suppresses metastasis of human lung adenocarcinoma in a murine model. We found that metastatic NSCLC cell lines H2030‐BrM3(K‐ras^G12C mutation) and PC9‐BrM3 (EGFR^Δexon19 mutation) had a significantly decreased p‐YAP(S127)/YAP ratio compared to parental H2030 (K‐ras^G12C mutation) and PC9 (EGFR^Δexon19 mutation) cells (P < .05). H2030‐BrM3 cells had significantly increased YAP mRNA and expression of Hippo downstream genes CTGF and CYR61 compared to parental H2030 cells (P < .05). Inhibition of YAP by short hairpin RNA (shRNA) and small interfering RNA (siRNA) significantly decreased mRNA expression in downstream genes CTGF and CYR61 in H2030‐BrM3 cells (P < .05). In addition, inhibiting YAP by YAP shRNA significantly decreased migration and invasion abilities of H2030‐BrM3 cells (P < .05). We are first to show that mice inoculated with YAP shRNA‐transfected H2030‐BrM3 cells had significantly decreased metastatic tumour burden and survived longer than control mice (P < .05). Collectively, our results suggest that YAP plays an important role in promoting lung adenocarcinoma brain metastasis and that direct inhibition of YAP by shRNA suppresses H2030‐BrM3 cell brain metastasis in a murine model.     

CD44+ fibroblasts increases breast cancer cell survival and drug resistance via IGF2BP3‐CD44‐IGF2 signalling

CD44, a cell adhesion protein, involves in various process in cancer such as cell survival and metastasis. Most researches on CD44 in cancer focus on cancer cells. Recently, it is found that CD44 expression is high in fibroblasts of tumour microenvironment. However, its role in communication between fibroblasts and breast cancer cells is seldom known. In this study, CD44‐positive (CD44⁺Fbs) and CD44‐negative carcinoma‐associated fibroblasts (CD44^?Fbs) were isolated and cocultured with breast cancer cells for analysis of cell survival and drug resistance. We found that CD44⁺Fbs promoted breast cancer cell survival and paclitaxel resistance and inhibited paclitaxel‐induced apoptosis. Our further research for the molecular mechanism showed that IGF2BP3 bound to CD44 mRNA and enhanced CD44 expression, which increased IGF2 levels of fibroblasts and then stimulated breast cancer cell proliferation and drug resistance. IGF2 was found to activate Hedgehog signal pathway in breast cancer cells. In conclusion, the results illustrated that in CD44⁺Fbs, binding of IGF2BP3 and CD44 promotes IGF2 expression and then accelerates breast cancer cell proliferation, survival and induced chemotherapy resistance likely by activating Hedgehog signal pathways.     

Epigenetic silencing of DACH1 induces the invasion and metastasis of gastric cancer by activating TGF‐β signalling

Gastric cancer (GC) is the fourth most common malignancy in males and the fifth most common malignancy in females worldwide. DACH1 is frequently methylated in hepatic and colorectal cancer. To further understand the regulation and mechanism of DACH1 in GC, eight GC cell lines, eight cases of normal gastric mucosa, 98 cases of primary GC and 50 cases of adjacent non‐tumour tissues were examined. Methylation‐specific PCR, western blot, transwell assay and xenograft mice were used in this study. Loss of DACH1 expression correlated with promoter region methylation in GC cells, and re‐expression was induced by 5‐Aza‐2′‐deoxyazacytidine. DACH1 is methylated in 63.3% (62/98) of primary GC and 38% (19/50) of adjacent non‐tumour tissues, while no methylation was found in normal gastric mucosa. Methylation of DACH1 correlated with reduced expression of DACH1 (P < 0.01), late tumour stage (stage III/IV) (P < 0.01) and lymph node metastasis (P < 0.05). DACH1 expression inhibited epithelial–mesenchymal transition and metastasis by inhibiting transforming growth factor (TGF)‐β signalling and suppressed GC cell proliferation through inducing G2/M phase arrest. The tumour size is smaller in DACH1‐expressed BGC823 cell xenograft mice than in unexpressed group (P < 0.01). Restoration of DACH1 expression also sensitized GC cells to docetaxel. These studies suggest that DACH1 is frequently methylated in human GC and expression of DACH1 was controlled by promoter region methylation. DACH1 suppresses GC proliferation, invasion and metastasis by inhibiting TGF‐β signalling pathways both in vitro and in vivo. Epigenetic silencing DACH1 may induce GC cells' resistance to docetaxel.     

miR‐655 suppresses epithelial‐to‐mesenchymal transition by targeting Prrx1 in triple‐negative breast cancer

Triple‐negative breast cancer (TNBC) is a highly aggressive breast cancer subtype that lacks effective targeted therapies. The epithelial‐to‐mesenchymal transition (EMT) is a key contributor in the metastatic process. In this study, we found that miR‐655 was down‐regulated in TNBC, and its expression levels were associated with molecular‐based classification and lymph node metastasis in breast cancer. These findings led us to hypothesize that miR‐655 overexpression may inhibit EMT and its associated traits of TNBC. Ectopic expression of miR‐655 not only induced the up‐regulation of cytokeratin and decreased vimentin expression but also suppressed migration and invasion of mesenchymal‐like cancer cells accompanied by a morphological shift towards the epithelial phenotype. In addition, we found that miR‐655 was negatively correlated with Prrx1 in cell lines and clinical samples. Overexpression of miR‐655 significantly suppressed Prrx1, as demonstrated by Prrx1 3′‐untranslated region luciferase report assay. Our study demonstrated that miR‐655 inhibits the acquisition of the EMT phenotype in TNBC by down‐regulating Prrx1, thereby inhibiting cell migration and invasion during cancer progression.     

Phospholipase C ε‐1 inhibits p53 expression in lung cancer

The pathogenesis of lung cancer is to be further investigated. Recent reports indicate that phospholipase C ε‐1 (PLCE1) is a critical molecule involved in tumour growth. This study aims to investigate the role of PLCE1 in the regulation of apoptosis in lung cancer cells. In this study, the surgically removed non‐small‐cell lung cancer (NSCLC) tissue was collected from 36 patients. Single NSCLC cells were prepared from the tissue, in which immune cells of CD3⁺, CD11c⁺, CD19⁺, CD68⁺ and CD14⁺ were eliminated by magnetic cell sorting. The expression of PLCE1 and p53 was assessed by quantitative real‐time polymerase chain reaction and Western blotting. Apoptosis of NSCLC cells was analysed by flow cytometry. The results showed that, in cultured NSCLC cells, high levels of PLCE1 and low levels p53 were detected; the two molecules showed a negative correlation (p < 0.01). The addition of anti‐PLCE1 antibody increased the expression of p53 in NSCLC cells, which increased the frequency of apoptotic NSCLC cells. We conclude that NSCLC cells express high levels of PLCE1, which suppresses the expression of p53 in NSCLC cells. PLCE1 can be a therapeutic target of NSCLC. Copyright © 2013 John Wiley & Sons, Ltd.     

miR‐515‐5p controls cancer cell migration through MARK4 regulation

Here, we show that miR‐515‐5p inhibits cancer cell migration and metastasis. RNA‐seq analyses of both oestrogen receptor receptor‐positive and receptor‐negative breast cancer cells overexpressing miR‐515‐5p reveal down‐regulation of NRAS, FZD4, CDC42BPA, PIK3C2B and MARK4 mRNAs. We demonstrate that miR‐515‐5p inhibits MARK4 directly 3′ UTR interaction and that MARK4 knock‐down mimics the effect of miR‐515‐5p on breast and lung cancer cell migration. MARK4 overexpression rescues the inhibitory effects of miR‐515‐5p, suggesting miR‐515‐5p mediates this process through MARK4 down‐regulation. Furthermore, miR‐515‐5p expression is reduced in metastases compared to primary tumours derived from both in vivo xenografts and samples from patients with breast cancer. Conversely, miR‐515‐5p overexpression prevents tumour cell dissemination in a mouse metastatic model. Moreover, high miR‐515‐5p and low MARK4 expression correlate with increased breast and lung cancer patients' survival, respectively. Taken together, these data demonstrate the importance of miR‐515‐5p/MARK4 regulation in cell migration and metastasis across two common cancers.     

DLG5 suppresses breast cancer stem cell‐like characteristics to restore tamoxifen sensitivity by inhibiting TAZ expression

Tamoxifen (TAM) is a primary drug for treatment of estrogen receptor positive breast cancer. However, TAM resistance remains a serious threat to breast cancer patients and may be attributed to increased stemness of breast cancer. Here, we show that discs large homolog 5 (DLG5) expression is down‐regulated in TAM‐resistant breast cancer and cells. DLG5 silencing decreased the sensitivity to TAM and increased the frequency and stemness of CD44⁺/CD24^? breast cancer stem cells (BCSCs) and TAZ, a transducer of the Hippo pathway, expression in MCF7 cells while DLG5 overexpression had opposite effects. TAZ silencing restored the sensitivity to TAM and reduced the frequency and stemness in TAM‐resistant breast cancer cells. Taken together, our data indicate that down‐regulated DLG5 expression increases the stemness of breast cancer cells by enhancing TAZ expression, contributing to TAM resistance in breast cancer.     

miR‐200c suppresses stemness and increases cellular sensitivity to trastuzumab in HER2+ breast cancer

Resistance to trastuzumab remains a major obstacle in HER2‐overexpressing breast cancer treatment. miR‐200c is important for many functions in cancer stem cells (CSCs), including tumour recurrence, metastasis and resistance. We hypothesized that miR‐200c contributes to trastuzumab resistance and stemness maintenance in HER2‐overexpressing breast cancer. In this study, we used HER2‐positive SKBR3, HER2‐negative MCF‐7, and their CD44⁺CD24^? phenotype mammospheres SKBR3‐S and MCF‐7‐S to verify. Our results demonstrated that miR‐200c was weakly expressed in breast cancer cell lines and cell line stem cells. Overexpression of miR‐200c resulted in a significant reduction in the number of tumour spheres formed and the population of CD44⁺CD24^? phenotype mammospheres in SKBR3‐S. Combining miR‐200c with trastuzumab can significantly reduce proliferation and increase apoptosis of SKBR3 and SKBR3‐S. Overexpression of miR‐200c also eliminated its downstream target genes. These genes were highly expressed and positively related in breast cancer patients. Overexpression of miR‐200c also improved the malignant progression of SKBR3‐S and SKBR3 in vivo. miR‐200c plays an important role in the maintenance of the CSC‐like phenotype and increases drug sensitivity to trastuzumab in HER2+ cells and stem cells.     

TLR5 is a new reporter for triple‐negative breast cancer indicated by radioimmunoimaging and fluorescent staining

Triple‐negative breast cancer (TNBC) is a highly aggressive tumour that lacks marker for targeted diagnosis. Recently, it was reported that toll‐like receptor 5 (TLR5) was associated with some kind of tumours, especially in TNBC, but whether it could be used as a non‐invasive monitoring target is not fully understood. Here, we established TLR5^? 4T1 cell line with lentivirus‐shRNA‐TLR5 knock‐down transfection (with tag GFP, green fluorescent protein, TLR5^? 4T1) and control TLR5⁺ 4T1 cell line with negative control lentivirus transfection. The effect of TLR5 down‐regulation was detected with qPCR and Western blot. ¹²⁵I‐anti‐TLR5 mAb and control isotype ¹²⁵I‐IgG were prepared and injected to TLR5^+/? 4T1‐bearing mice models, respectively. Whole‐body phosphor‐autoradiography, fluorescence imaging and biodistribution were performed. Furthermore, ex vivo tumour TLR5 expression was proved through immunohistochemistry staining. We found that ¹²⁵I‐anti‐TLR5 mAb could bind to TLR5⁺ 4T1 with high affinity and specificity. Whole‐body phosphor‐autoradiography after ¹²⁵I‐anti‐TLR5 mAb injection showed TLR5⁺ 4T1 tumour images in 24 hours, more clearly in 48 hours. Radioactivities in tumour tissues were positively related with TLR5 expression. Biodistribution assay showed that ¹²⁵I‐anti‐TLR5 mAb was mainly metabolized through the liver and kidney, and ¹²⁵I‐anti‐TLR5 mAb was much more accumulated in TLR5⁺ 4T1 tumour than TLR5^? 4T1. In vivo fluorescence imaging successfully showed tumour tissues clearly both in TLR5⁺ and TLR5^? 4T1 mice compared with lentivirus untreated 4T1 tumour. Immunohistochemistry staining showed that TLR5 expression in tumours was indeed down‐regulated in TLR5^? 4T1 mice. Our results indicated that ¹²⁵I‐antiTLR5 mAb was an ideal agent for non‐invasive imaging of TLR5⁺ tumours; TLR5 may be as a novel molecular target for TNBC non‐invasive diagnosis.     

Enhanced immune response induced by P5 HER2/neu‐derived peptide‐pulsed dendritic cells as a preventive cancer vaccine

Dendritic cells are special and powerful antigen‐presenting cells that can induce primary immune responses against tumour‐associated antigens. They can present antigens via both MHC‐I and MHC‐II, so they have the ability to stimulate both cytotoxic T lymphocytes and T helper cells. Furthermore, CD8⁺ cytotoxic T lymphocytes require activation by CD4⁺ T cells. This requires a CD4⁺T cell activator molecule, of which PADRE is one of the best. We chose an approach to use both of these important arms of the immune system. We prepared dendritic cells from mouse bone marrow, loaded them with our target peptides (P5 peptide alone or P5 + PADRE), and then injected these pulsed dendritic cells alone or in combination with CpG‐ODN (as adjuvant) into BALB/C mice. After the last boosting dose, mice were inoculated with TUBO cells, which overexpress HER2/neu. Two weeks after the tumour cell injection, immunological tests were performed on splenocyte suspensions, and the remaining mice were evaluated for tumour growth and survival. Our data indicate the formulation that contains PADRE plus P5 loaded onto DC in combination with CpG‐ODN was the most effective formulation at inducing immune responses. Interferon production in CD4⁺ and CD8⁺ gated cells, cytotoxicity rates of target cells and mice survival were all significantly greater in this group than in controls, and all the mice in this group were tumour‐free throughout the experiment. Based on our results and the role of HER2/neu as a candidate in human immunotherapy, this approach may be an effective cancer treatment.     

Influence of type‐I Interferon receptor expression level on the response to type‐I Interferons in human pancreatic cancer cells

Pancreatic cancer is a highly aggressive malignancy with limited treatment options. Type‐I interferons (e.g. IFN‐α/‐β) have several anti‐tumour activities. Over the past few years, clinical studies evaluating the effect of adjuvant IFN‐α therapy in pancreatic cancer yielded equivocal results. Although IFN‐α and ‐β act via the type‐I IFN receptor, the role of the number of receptors present on tumour cells is still unknown. Therefore, this study associated, for the first time, in a large panel of pancreatic cancer cell lines the effects of IFN‐α/‐β with the expression of type‐I IFN receptors. The anti‐tumour effects of IFN‐α or IFN‐β on cell proliferation and apoptosis were evaluated in 11 human pancreatic cell lines. Type‐I IFN receptor expression was determined on both the mRNA and protein level. After 7 days of incubation, IFN‐α significantly reduced cell growth in eight cell lines by 5–67%. IFN‐β inhibited cell growth statistically significant in all cell lines by 43–100%. After 3 days of treatment, IFN‐β induced significantly more apoptosis than IFN‐α. The cell lines variably expressed the type‐I IFN receptor. The maximal inhibitory effect of IFN‐α was positively correlated with the IFNAR‐1 mRNA (P < 0.05, r = 0.63), IFNAR‐2c mRNA (P < 0.05, r = 0.69) and protein expression (P < 0.05, r = 0.65). Human pancreatic cancer cell lines variably respond to IFN‐α and ‐β. The expression level of the type‐I IFN receptor is of predictive value for the direct anti‐tumour effects of IFN‐α treatment. More importantly, IFN‐β induces anti‐tumour effects already at much lower concentrations, is less dependent on interferon receptor expression and seems, therefore, more promising than IFN‐α.