Small interfering RNA library screen identified polo-like kinase-1 (PLK1) as a potential therapeutic target for breast cancer that uniquely eliminates tumor-initiating cells

Introduction

Triple-negative breast cancer (TNBC) high rate of relapse is thought to be due to the presence of tumor-initiating cells (TICs), molecularly defined as being CD44^high/CD24^-/low. TICs are resilient to chemotherapy and radiation. However, no currently accepted molecular target exists against TNBC and, moreover, TICs. Therefore, we sought the identification of kinase targets that inhibit TNBC growth and eliminate TICs.

Methods

A genome-wide human kinase small interfering RNA (siRNA) library (691 kinases) was screened against the TNBC cell line SUM149 for growth inhibition. Selected siRNAs were then tested on four different breast cancer cell lines to confirm the spectrum of activity. Their effect on the CD44^high subpopulation and sorted CD44^high/CD24^-/low cells of SUM149 also was studied. Further studies were focused on polo-like kinase 1 (PLK1), including its expression in breast cancer cell lines, effect on the CD44^high/CD24^-/low TIC subpopulation, growth inhibition, mammosphere formation, and apoptosis, as well as the activity of the PLK1 inhibitor, BI 2536.

Results

Of the 85 kinases identified in the screen, 28 of them were further silenced by siRNAs on MDA-MB-231 (TNBC), BT474-M1 (ER⁺/HER2⁺, a metastatic variant), and HR5 (ER⁺/HER2⁺, a trastuzumab-resistant model) cells and showed a broad spectrum of growth inhibition. Importantly, 12 of 28 kinases also reduced the CD44^high subpopulation compared with control in SUM149. Further tests of these 12 kinases directly on a sorted CD44^high/CD24^-/low TIC subpopulation of SUM149 cells confirmed their effect. Blocking PLK1 had the greatest growth inhibition on breast cancer cells and TICs by about 80% to 90% after 72 hours. PLK1 was universally expressed in breast cancer cell lines, representing all of the breast cancer subtypes, and was positively correlated to CD44. The PLK1 inhibitor BI 2536 showed similar effects on growth, mammosphere formation, and apoptosis as did PLK1 siRNAs. Finally, whereas paclitaxel, doxorubicin, and 5-fluorouracil enriched the CD44^high/CD24^-/low population compared with control in SUM149, subsequent treatment with BI 2536 killed the emergent population, suggesting that it could potentially be used to prevent relapse.

Conclusion

Inhibiting PLK1 with siRNA or BI 2536 blocked growth of TNBCs including the CD44^high/CD24^-/low TIC subpopulation and mammosphere formation. Thus, PLK1 could be a potential therapeutic target for the treatment of TNBC as well as other subtypes of breast cancer.     

CD44+/CD24− breast cancer cells isolated from MCF-7 cultures exhibit enhanced angiogenic properties

Background

Recent studies suggest that the relationship between cancer stem cells (CSCs) and the vascular niche may be bidirectional; the niche can support the growth and renewal of CSCs, and CSCs may contribute to the maintenance of the niche. There is little knowledge concerning the role of breast cancer stem cells in promoting tumor angiogenesis.

Aim

For human breast cancers, CSCs have been shown to be associated with a CD44⁺/CD24 ^? phenotype. We investigated the potential activities of CD44⁺/CD24 ^? breast cancer stem cells in promoting tumor angiogenesis.

Methods

The expression of pro-angiogenic genes was determined by quantitative real-time RT-PCR. Endothelial cell migration assays were employed to evaluate effects of conditioned media from CD44⁺/CD24 ^? on human umbilical vein endothelial cells. A chorioallantoic membrane (CAM) assay was used to study the potential of CD44⁺/CD24 ^? cells to promote angiogenesis.

Results

In our study, CD44⁺/CD24 ^? cells expressed elevated levels of pro-angiogenic factors compared with CD44⁺/CD24⁺ cells. CD44⁺/CD24 ^? cell-conditioned media significantly increased endothelial cell migration. Breast cancer cell lines enriched with CD44⁺/CD24 ^? cells were more pro-angiogenic in the CAM assay than those lacking a CD44⁺/CD24 ^? subpopulation. CD44⁺/CD24 ^? cells sorted from MCF-7 cell lines were more pro-angiogenic in a CAM assay than CD44⁺/CD24⁺ cells. Furthermore, the VEGF concentration was significantly higher in CD44⁺/CD24 ^? cell-conditioned media than in CD44⁺/CD24⁺ cell-conditioned media. The pro-angiogenic effect of CD44⁺/CD24 ^? cells on endothelial cells was abolished by bevacizumab.

Conclusion

Our findings demonstrate that CD44⁺/CD24 ^? breast cancer stem cells have substantial pro-angiogenic potential and activity. This provides new insights to explore in the development of targeted therapies.     

Microarray-based analysis of microRNA expression in breast cancer stem cells

Background

This study aimed to determine the miRNA profile in breast cancer stem cells (BCSCs) and to explore the functions of characteristic BCSC miRNAs.

Methods

We isolated ESA⁺CD44⁺CD24^-/low BCSCs from MCF-7 cells using fluorescence-activated cell sorting (FACS). A human breast cancer xenograft assay was performed to validate the stem cell properties of the isolated cells, and microarray analysis was performed to screen for BCSC-related miRNAs. These BCSC-related miRNAs were selected for bioinformatic analysis and target prediction using online software programs.

Results

The ESA⁺CD44⁺CD24^-/low cells had up to 100- to 1000-fold greater tumor-initiating capability than the MCF-7 cells. Tumors initiated from the ESA⁺CD44⁺CD24^-/low cells were included of luminal epithelial and myoepithelial cells, indicating stem cell properties. We also obtained miRNA profiles of ESA⁺CD44⁺CD24^-/low BCSCs. Most of the possible targets of potential tumorigenesis-related miRNAs were oncogenes, anti-oncogenes or regulatory genes.

Conclusions

We identified a subset of miRNAs that were differentially expressed in BCSCs, providing a starting point to explore the functions of these miRNAs. Evaluating characteristic BCSC miRNAs represents a new method for studying breast cancer-initiating cells and developing therapeutic strategies aimed at eradicating the tumorigenic subpopulation of cells in breast cancer.     

Brca1 breast tumors contain distinct CD44+/CD24- and CD133+ cells with cancer stem cell characteristics

Introduction

Whether cancer stem cells occur in BRCA1-associated breast cancer and contribute to therapeutic response is not known.

Methods

We generated and characterized 16 cell lines from five distinct Brca1deficient mouse mammary tumors with respect to their cancer stem cell characteristics.

Results

All cell lines derived from one tumor included increased numbers of CD44⁺/CD24^- cells, which were previously identified as human breast cancer stem cells. All cell lines derived from another mammary tumor exhibited low levels of CD44⁺/CD24^- cells, but they harbored 2% to 5.9% CD133⁺ cells, which were previously associated with cancer stem cells in other human and murine tumors. When plated in the absence of attachment without presorting, only those cell lines that were enriched in either stem cell marker formed spheroids, which were further enriched in cells expressing the respective cancer stem cell marker. In contrast, cells sorted for CD44⁺/CD24^- or CD133⁺ markers lost their stem cell phenotype when cultured in monolayers. As few as 50 to 100 CD44⁺/CD24^- or CD133⁺ sorted cells rapidly formed tumors in nonobese diabetic/severe combined immunodeficient mice, whereas 50-fold to 100-fold higher numbers of parental or stem cell depleted cells were required to form few, slow-growing tumors. Expression of stem cell associated genes, including Oct4, Notch1, Aldh1, Fgfr1, and Sox1, was increased in CD44⁺/CD24^- and CD133⁺ cells. In addition, cells sorted for cancer stem cell markers and spheroid-forming cells were significantly more resistant to DNA-damaging drugs than were parental or stem cell depleted populations, and they were sensitized to the drugs by the heat shock protein-90 inhibitor 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride).

Conclusion

Brca1-deficient mouse mammary tumors harbor heterogeneous cancer stem cell populations, and CD44⁺/CD24^- cells represent a population that correlates with human breast cancer stem cells.     

Pharmacological inhibition of AKT sensitizes MCF-7 human breast cancer-initiating cells to radiation

Background

Caner-initiating cells (CICs or cancer stem cells) have been shown both experimentally and clinically to be resistant to radiation. The mechanism underlying radioresistance remains unclear.

Methods

In the present study, we screened 51 genes which are potentially important in mediating radioresistance of breast CICs.

Results

The expression of AKT1 and AKT2 at protein and mRNA levels was dramatically increased among the screened genes by 8 Gy radiation treatment in MCF-7 mammosphere cells (predominantly CD24^–/low/CD44⁺ CICs), but not in the bulk population of MCF-7 cells (predominantly CD24⁺/CD44⁺). Using apoptosis and clonogenic survival assays, we found pharmacological inhibition of AKT with selective inhibitors of AKT sensitized MCF-7 mammosphere cells, but not MCF-7 monolayer cells to radiation.

Conclusion

The present findings suggest that treatment with AKT inhibitors prior to ionizing radiation treatment may be a potential benefit to patients with breast cancer, in particular to eradiate breast CICs.     

Can CD44+/CD24- Tumor Cells Be Used to Determine the Extent of Breast Cancer Invasion Following Neoadjuvant Chemotherapy?

Purpose

To investigate the distribution of CD44⁺/CD24^- cells in breast cancers in relation to tumor size before and after the administration of neoadjuvant chemotherapy.

Methods

CD44⁺/CD24^- tumor cells obtained from breast cancer specimens were characterized in vivo and in vitro using tumor formation assays and mammosphere generation assays, respectively. The distribution of CD44+/CD24- tumor cells in 78 breast cancer specimens following administration of neoadjuvant chemotherapy was also evaluated using immunofluorescence assays, and this distribution was compared with the extent of tumor invasion predicted by Response Evaluation Criteria in Solid Tumours (RECIST).

Results

In 27/78 cases, complete remission (CR) was identified using RECIST. However, 18 of these CR cases were associated with a scattered distribution of tumor stem cells in the outline of the original tumor prior to neoadjuvant chemotherapy. After neoadjuvant chemotherapy, 24 cases involved cancer cells that were confined to the tumor outline, and 21 cases had tumor cells or tumor stem cells overlapping the tumor outline. In addition, there were 6 patients who were insensitive to chemotherapy, and in these cases, both cancer cells and stem cells were detected outside the contours of the tumor volume imaged prior to chemotherapy.

Conclusion

CD44+/CD24- tumor cells may be an additional parameter to evaluate when determining the extent of breast cancer invasion.     

Hypoxia regulates stemness of breast cancer MDA-MB-231 cells

Human breast cancers include cancer stem cell populations as well as non-tumorigenic cancer cells. Breast cancer stem cells possess self-renewal capability and thus are the root cause of recurrence and metastasis of malignant tumors. Hypoxia is a fundamental pathological feature of solid tumor tissues and exerts a wide range of effects on the biological behavior of cancer cells. However, there is little information on the role of hypoxia in modulating the stemness of breast cancer cells. In the present study, we cultured MDA-MB-231 cells in a hypoxic gas mixture to simulate the hypoxic environment in tissues and to determine how hypoxia conditions could affect the cell proliferation, apoptosis, cytotoxicity, and colony-forming ability. Expression of the stem cell phenotype CD24^?CD44⁺ESA⁺ was analyzed to assess the effects of hypoxia on stemness transformation in MDA-MB-231 cells. Our results found that the cell toxicity of MDA-MB-231 cells was not affected by hypoxia. Hypoxia could slightly inhibit the growth of MDA-MB-231 cells, but the inhibitory effect is not significant when compared with normoxic control. Moreover, hypoxia significantly blocked the apoptosis in MDA-MB-231 cells (P < 0.05). The proportion of CD24^?CD44⁺ESA⁺ cells in MDA-MB-231 cells was increased greatly after they were treated with hypoxia, and cell colony-formation rate of MDA-MB-231 cells also increased significantly in hypoxia-treated cells. These results encourage the exploration of hypoxia as a mechanism which might not be underestimated in chemo-resistant breast cancer treatment.     

Clonal relatedness between lobular carcinoma in situ and synchronous malignant lesions

Introduction

Mammary-specific overexpression of Six1 in mice induces tumors that resemble human breast cancer, some having undergone epithelial to mesenchymal transition (EMT) and exhibiting stem/progenitor cell features. Six1 overexpression in human breast cancer cells promotes EMT and metastatic dissemination. We hypothesized that Six1 plays a role in the tumor initiating cell (TIC) population specifically in certain subtypes of breast cancer, and that by understanding its mechanism of action, we could potentially develop new means to target TICs.

Methods

We examined gene expression datasets to determine the breast cancer subtypes with Six1 overexpression, and then examined its expression in the CD24^low/CD44⁺ putative TIC population in human luminal breast cancers xenografted through mice and in luminal breast cancer cell lines. Six1 overexpression, or knockdown, was performed in different systems to examine how Six1 levels affect TIC characteristics, using gene expression and flow cytometric analysis, tumorsphere assays, and in vivo TIC assays in immunocompromised and immune-competent mice. We examined the molecular pathways by which Six1 influences TICs using genetic/inhibitor approaches in vitro and in vivo. Finally, we examined the expression of Six1 and phosphorylated extracellular signal-regulated kinase (p-ERK) in human breast cancers.

Results

High levels of Six1 are associated with adverse outcomes in luminal breast cancers, particularly the luminal B subtype. Six1 levels are enriched in the CD24^low/CD44⁺ TIC population in human luminal breast cancers xenografted through mice, and in tumorsphere cultures in MCF7 and T47D luminal breast cancer cells. When overexpressed in MCF7 cells, Six1expands the TIC population through activation of transforming growth factor-beta (TGF-β) and mitogen activated protein kinase (MEK)/ERK signaling. Inhibition of ERK signaling in MCF7-Six1 cells with MEK1/2 inhibitors, U0126 and AZD6244, restores the TIC population of luminal breast cancer cells back to that observed in control cells. Administration of AZD6244 dramatically inhibits tumor formation efficiency and metastasis in cells that express high levels of Six1 ectopically or endogenously. Finally, we demonstrate that Six1 significantly correlates with phosphorylated ERK in human breast cancers.

Conclusions

Six1 plays an important role in the TIC population in luminal breast cancers and induces a TIC phenotype by enhancing both TGF-β and ERK signaling. MEK1/2 kinase inhibitors are potential candidates for targeting TICs in breast tumors.     

Cross-priming of cyclin B1, MUC-1 and survivin-specific CD8+ T cells by dendritic cells loaded with killed allogeneic breast cancer cells

Introduction

The ability of dendritic cells (DCs) to take up whole tumor cells and process their antigens for presentation to T cells ('cross-priming') is an important mechanism for induction of tumor specific immunity.

Methods

In vitro generated DCs were loaded with killed allogeneic breast cancer cells and offered to autologous naïve CD8⁺ T cells in 2-week and/or 3-week cultures. CD8⁺ T cell differentiation was measured by their capacity to secrete effector cytokines (interferon-γ) and kill breast cancer cells. Specificity was measured using peptides derived from defined breast cancer antigens.

Results

We found that DCs loaded with killed breast cancer cells can prime naïve CD8⁺ T cells to differentiate into effector cytotoxic T lymphocytes (CTLs). Importantly, these CTLs primed by DCs loaded with killed HLA-A*0201^- breast cancer cells can kill HLA-A*0201⁺ breast cancer cells. Among the tumor specific CTLs, we found that CTLs specific for HLA-A2 restricted peptides derived from three well known shared breast tumor antigens, namely cyclin B1, MUC-1 and survivin.

Conclusion

This ability of DCs loaded with killed allogeneic breast cancer cells to elicit multiantigen specific immunity supports their use as vaccines in patients with breast cancer.     

Deoxycholate promotes survival of breast cancer cells by reducing the level of pro-apoptotic ceramide

Introduction

At physiologic concentration in serum, the bile acid sodium deoxycholate (DC) induces survival and migration of breast cancer cells. Here we provide evidence of a novel mechanism by which DC reduces apoptosis that is induced by the sphingolipid ceramide in breast cancer cells.

Methods

Murine mammacarcinoma 4T1 cells were used in vitro to determine apoptosis and alteration of sphingolipid metabolism by DC, and in vivo to quantify the effect of DC on metastasis.

Results

We found that DC increased the number of intestinal metastases generated from 4T1 cell tumors grafted into the fat pad. The metastatic nodes contained slowly dividing cancer cells in immediate vicinity of newly formed blood vessels. These cells were positive for CD44, a marker that has been suggested to be expressed on breast cancer stem cells. In culture, a subpopulation (3 ± 1%) of slowly dividing, CD44⁺ cells gave rise to rapidly dividing, CD44^- cells. DC promoted survival of CD44⁺ cells, which was concurrent with reduced levels of activated caspase 3 and ceramide, a sphingolipid inducing apoptosis in 4T1 cells. Z-guggulsterone, an antagonist of the farnesoid-X-receptor, obliterated this anti-apoptotic effect, indicating that DC increased cell survival via farnesoid-X-receptor. DC also increased the gene expression of the vascular endothelial growth factor receptor 2 (Flk-1), suggesting that DC enhanced the initial growth of secondary tumors adjacent to blood vessels. The Flk-1 antagonist SU5416 obliterated the reduction of ceramide and apoptosis by DC, indicating that enhanced cell survival is due to Flk-1-induced reduction in ceramide.

Conclusions

Our findings show, for the first time, that DC is a natural tumor promoter by elevating Flk-1 and decreasing ceramide-mediated apoptosis of breast cancer progenitor cells. Reducing the level or effect of serum DC and elevating ceramide in breast cancer progenitor cells by treatment with Z-guggulsterone and/or vascular endothelial growth factor receptor 2/Flk-1 antagonists may thus be a promising strategy to reduce breast cancer metastasis.     

Nuclear β-catenin and CD44 upregulation characterize invasive cell populations in non-aggressive MCF-7 breast cancer cells

Background

In breast cancer cells, the metastatic cell state is strongly correlated to epithelial-to-mesenchymal transition (EMT) and the CD44⁺/CD24^- stem cell phenotype. However, the MCF-7 cell line, which has a luminal epithelial-like phenotype and lacks a CD44⁺/CD24^- subpopulation, has rare cell populations with higher Matrigel invasive ability. Thus, what are the potentially important differences between invasive and non-invasive breast cancer cells, and are the differences related to EMT or CD44/CD24 expression?

Methods

Throughout the sequential selection process using Matrigel, we obtained MCF-7-14 cells of opposite migratory and invasive capabilities from MCF-7 cells. Comparative analysis of epithelial and mesenchymal marker expression was performed between parental MCF-7, selected MCF-7-14, and aggressive mesenchymal MDA-MB-231 cells. Furthermore, using microarray expression profiles of these cells, we selected differentially expressed genes for their invasive potential, and performed pathway and network analysis to identify a set of interesting genes, which were evaluated by RT-PCR, flow cytometry or function-blocking antibody treatment.

Results

MCF-7-14 cells had enhanced migratory and invasive ability compared with MCF-7 cells. Although MCF-7-14 cells, similar to MCF-7 cells, expressed E-cadherin but neither vimentin nor fibronectin, β-catenin was expressed not only on the cell membrane but also in the nucleus. Furthermore, using gene expression profiles of MCF-7, MCF-7-14 and MDA-MB-231 cells, we demonstrated that MCF-7-14 cells have alterations in signaling pathways regulating cell migration and identified a set of genes (PIK3R1, SOCS2, BMP7, CD44 and CD24). Interestingly, MCF-7-14 and its invasive clone CL6 cells displayed increased CD44 expression and downregulated CD24 expression compared with MCF-7 cells. Anti-CD44 antibody treatment significantly decreased cell migration and invasion in both MCF-7-14 and MCF-7-14 CL6 cells as well as MDA-MB-231 cells.

Conclusions

MCF-7-14 cells are a novel model for breast cancer metastasis without requiring constitutive EMT and are categorized as a "metastable phenotype", which can be distinguished from both epithelial and mesenchymal cells. The alterations and characteristics of MCF-7-14 cells, especially nuclear β-catenin and CD44 upregulation, may characterize invasive cell populations in breast cancer.

     

Gene set enrichment analysis provides insight into novel signalling pathways in breast cancer stem cells

Background:

Tumour-initiating cells (TICs) or cancer stem cells can exist as a small population in malignant tissues. The signalling pathways activated in TICs that contribute to tumourigenesis are not fully understood.

Methods:

Several breast cancer cell lines were sorted with CD24 and CD44, known markers for enrichment of breast cancer TICs. Tumourigenesis was analysed using sorted cells and total RNA was subjected to gene expression profiling and gene set enrichment analysis (GSEA).

Results:

We showed that several breast cancer cell lines have a small population of CD24^−/low/CD44⁺ cells in which TICs may be enriched, and confirmed the properties of TICs in a xenograft model. GSEA revealed that CD24^−/low/CD44⁺ cell populations are enriched for genes involved in transforming growth factor-β, tumour necrosis factor, and interferon response pathways. Moreover, we found the presence of nuclear factor-κB (NF-κB) activity in CD24^−/low/CD44⁺ cells, which was previously unrecognised. In addition, NF-κB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) prevented tumourigenesis of CD24^−/low/CD44⁺ cells in vivo.

Conclusion:

Our findings suggest that signalling pathways identified using GSEA help to identify molecular targets and biomarkers for TIC-like cells.     

A mammosphere formation RNAi screen reveals that ATG4A promotes a breast cancer stem-like phenotype

Introduction

Breast cancer stem cells are suspected to be responsible for tumour recurrence, metastasis formation as well as chemoresistance. Consequently, great efforts have been made to understand the molecular mechanisms underlying cancer stem cell maintenance. In order to study these rare cells in-vitro, they are typically enriched via mammosphere culture. Here we developed a mammosphere-based negative selection shRNAi screening system suitable to analyse the involvement of thousands of genes in the survival of cells with cancer stem cell properties.

Methods

We describe a sub-population expressing the stem-like marker CD44⁺/CD24^-/low in SUM149 that were enriched in mammospheres. To identify genes functionally involved in the maintenance of the sub-population with cancer stem cell properties, we targeted over 5000 genes by RNAi and tested their ability to grow as mammospheres. The identified candidate ATG4A was validated in mammosphere and soft agar colony formation assays. Further, we evaluated the influence of ATG4A expression on the sub-population expressing the stem-like marker CD44⁺/CD24^low. Next, the tumorigenic potential of SUM149 after up- or down-regulation of ATG4A was examined by xenograft experiments.

Results

Using this method, Jak-STAT as well as cytokine signalling were identified to be involved in mammosphere formation. Furthermore, the autophagy regulator ATG4A was found to be essential for the maintenance of a sub-population with cancer stem cell properties and to regulate breast cancer cell tumourigenicity in vivo.

Conclusion

In summary, we present a high-throughput screening system to identify genes involved in cancer stem cell maintenance and demonstrate its utility by means of ATG4A.     

The clinical effects of DC-CIK cells combined with chemotherapy in the treatment of advanced NSCLC

Objective

The aim of the study was to evaluate the safety and therapeutic effects of autologous dendritic cells co-cultured with cytokine-induced killer cells (DC-CIK) combined with chemotherapy in advanced non-small cell lung cancer (NSCLC) patients.

Methods

Fifty patients with advanced NSCLC (stages III to IV), who had received therapies in our Center (Department of Biotherapy, Affiliated to Cancer Hospital of Shanxi Medical University, Taiyuan, China) from August 2008 to January 2010, were treated by DC-CIK + chemotherapy as the combined treatment group; fifty advanced NSCLC patients treated with chemotherapy at the same time served as controls. The immunologic function, short-term therapeutic effects, the 1-year survival rate, the life quality, the chemotherapy side effects were compared between the two groups, the safety and therapeutic effects of DC-CIK cells therapy were observed too.

Results

There was no obvious change of subsets of T cells in peripheral blood before and after therapy in DC-CIK + chemotherapy group, and IFN-?? was improved after therapy in this group (P < 0.05); in chemotherapy alone group, the ratios of CD3⁺CD4⁺, CD3⁺CD8⁺, CD3^?CD56⁺ cells and the secretion of IL-2, TNF-?? decreased significantly after therapy (P < 0.05); the ratios of CD3⁺CD8⁺, CD3⁺CD56⁺ were improved after cell culture (P < 0.05). The disease control rate (DCR) of DC-CIK + chemotherapy group was higher than that in the chemotherapy alone group (78.0% vs 56.0%, P < 0.05); the 1-year survival rates of DC-CIK + chemotherapy group and chemotherapy alone group were 50% and 44% respectively, had no significant difference. Compared with chemotherapy alone group, the occurrence of chemotherapy side effects (including bone marrow suppression, nausea and vomiting, peripheral nerve toxicity) was less in the DC-CIK + chemotherapy group (P < 0.05). The physical and appetite were better in DC-CIK + chemotherapy group after therapy.

Conclusion

To compare with simple chemotherapy, DC-CIK + chemotherapy for advanced NSCLC is safe and effective, and it can improve patients?? life quality and remission rate, and prolong their survival time.     

Suppression of breast cancer cell growth by Na+/H+ exchanger regulatory factor 1 (NHERF1)

Introduction

Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1, also known as EBP50 or NHERF) is a putative tumour suppressor gene in human breast cancer. Located at 17q25.1, NHERF1 is frequently targeted during breast tumourigenesis. Loss of heterozygosity (LOH) at the NHERF1 locus is found in more than 50% of breast tumours. In addition, NHERF1 is mutated in a subset of primary breast tumours and breast cancer cell lines. LOH at the NHERF1 locus is strongly associated with aggressive features of breast tumours, implicating NHERF1 as a haploinsufficiency tumour suppressor gene. However, the putative NHERF1 tumour suppressor activity has not been functionally verified.

Methods

To confirm the NHERF1 tumour suppressor activity suggested by our genetic analyses, we used retrovirus-transduced short hairpin RNA (shRNA) to knock down NHERF1 expression in breast cancer cell lines MCF7 and T47D. These cells were then assessed for cell growth in vitro and in vivo. The control and NHERF1 knockdown cells were also serum-starved and re-fed to compare their cell cycle progression as measured by fluorescence-activated cell sorting analyses.

Results

We found that downregulation of the endogenous NHERF1 in T47D or MCF7 cells resulted in enhanced cell proliferation in both anchorage-dependent and -independent conditions compared with that of the vector control cells. NHERF1 knockdown T47D cells implanted at mammary fat pads of athymic mice formed larger tumours than did control cells. We found that serum-starved NHERF1 knockdown cells had a faster G₁-to-S transition after serum re-stimulation than the control cells. Immunoblotting showed that the accelerated cell cycle progression in NHERF1 knockdown cells was accompanied by increased expression of cyclin E and elevated Rb phosphorylation level.

Conclusion

Our findings suggested that the normal NHERF1 function in mammary epithelial cells involves blockage of cell cycle progression. Our study affirmed the tumour suppressor activity of NHERF1 in breast which may be related to its regulatory effect on cell cycle. It warrants future investigation of this novel tumour suppressor pathway in human breast cancer which may turn up therapeutic opportunities.     

Tumour-initiating capacity is independent of epithelial–mesenchymal transition status in breast cancer cell lines

Background:

Epithelial–mesenchymal transition (EMT) and cancer stem cells (CSCs) are considered to be crucial for cancer biology. The purpose of this study was to determine whether EMT directly led to the acquisition of tumour-initiating capacity in breast cancer cell lines.

Methods:

Epithelial–mesenchymal transition was induced in five breast cancer cell lines and one normal breast cell line by EMT-related cytokine stimulation. Mesenchymal–epithelial transition (MET) was induced by stably overexpressing miR-200c in three mesenchymal-like breast cancer cell lines. Molecular expression and cell function analysis were performed to evaluate the effect of EMT or MET on tumour-initiating capacity and other biological characteristics.

Results:

The induction of EMT did not enhance tumour-initiating capacity but, instead, conferred a CD44⁺/CD24^−/low phenotype as well as cell proliferation, migration, and resistance to doxorubicin and radiation on breast cancer cell lines. Furthermore, MET did not lead to inhibition or loss of the tumour-initiating capacity in mesenchymal-like breast cancer cell lines, but it markedly attenuated other malignant properties, including proliferation, invasion, and resistance to therapy.

Conclusions:

Epithelial–mesenchymal transition does not alter tumour-initiating capacity of breast cancer cells but some other biological characteristics. Therefore, EMT and tumour-initiating capacity may not be directly linked in breast cancer cell lines.     

The correlation of CD19 + CD24 + CD38 + B cells and other clinicopathological variables with the proportion of circulating Tregs in breast cancer patients

Background

T regulatory cells (Tregs) are known to negatively control immune response. The frequency of these cells was inversely correlated with clinical outcomes of breast cancer. CD19⁺CD24^hiCD38^hi cells also play a critical role in inflammation and autoimmune disease. However, their function in tumor immune response is less studied. In this study we aimed to determine the role of CD19⁺CD24^hiCD38^hi cells and some other clinicopathological variables in increasing the proportion of Tregs in breast cancer patients.

Methods

We selected 47 patients with invasive ductal breast carcinoma and 50 healthy controls and obtained their blood samples.

Results

The proportion of circulating CD4⁺CD25⁺Foxp3⁺ Tregs and CD19⁺CD24^hiCD38^hi cells was significantly increased in breast cancer patients. We also found that increased proportion of Tregs in breast cancer is correlated with HER2 amplification, advanced clinical stages, serum TGF-β1 and increased CD19⁺CD24^hiCD38^hi cells in the peripheral blood.

Conclusion

Altogether, our data suggest that as much as Tregs, CD19⁺CD24^hiCD38^hi B cells could also have a part in the suppression of immune response in breast cancer.

     

Suppression of myeloid cell leukemia-1 (Mcl-1) enhances chemotherapy-associated apoptosis in gastric cancer cells

Background

Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic protein that regulates apoptosis sensitivity in a variety of cell types. Here we evaluate the roles of Mcl-1 in chemotherapy-associated apoptosis in gastric cancer cells. In addition, our study examined whether Mcl-1 contributed to apoptosis resistance in so-called cancer stem cell (CSC)-like populations in gastric cancer.

Methods

Seven gastric cancer cell lines were used. The expression of Mcl-1 was assessed by either real-time polymerase chain reaction or Western blot analysis. Apoptosis was quantitated by morphological observation and caspase activity measurement. Adenovirus-mediated RNA interference (RNAi) technology was used to knockdown the expression of Mcl-1. The release of cytochrome c was evaluated by subcellular fractionation and immunoblot analysis. To identify and isolate the CSC-like populations, we used the CSC-associated cell surface marker CD44 and flow cytometry.

Results

Six out of the 7 gastric cancer cell lines overexpressed Mcl-1 protein. These Mcl-1-expressing cell lines were relatively resistant to chemotherapeutic agents such as 5-fluorouracil (5-FU) and cisplatin (CDDP). Depletion of Mcl-1 protein by RNAi technology effectively sensitized the cells to anticancer drug-induced mitochondrial cytochrome c release, caspase activation, and apoptosis. In addition, vast amounts of Mcl-1 mRNA were expressed in CD44-positive CSC-like cells. Mcl-1 suppression enhanced the apoptosis in CD44-positive cells to a level equivalent to that in CD44-negative cells, suggesting that Mcl-1 mediates chemotherapy resistance in CSC-like populations.

Conclusion

These results suggest that Mcl-1 mediates the resistance to apoptosis in gastric cancer cells by blocking the mitochondrial pathway of cell death. Mcl-1 depletion appears to be an attractive strategy to overcome chemotherapy resistance in gastric cancer cells.     

Breast cancer stromal fibroblasts promote the generation of CD44+CD24- cells through SDF-1/CXCR4 interaction

Background

Breast cancer stem cells (BCSCs) have been recently identified in breast carcinoma as CD44⁺CD24^- cells, which exclusively retain tumorigenic activity and display stem cell-like properties. Using a mammosphere culture technique, MCF7 mammosphere cells are found to enrich breast cancer stem-like cells expressing CD44⁺CD24^-. The stromal cells are mainly constituted by fibroblasts within a breast carcinoma, yet little is known of the contributions of the stromal cells to BCSCs.

Methods

Carcinoma-associated fibroblasts (CAFs) and normal fibroblasts (NFs) were isolated and identified by immunohistochemistry. MCF7 mammosphere cells were co-cultured with different stromal fibroblasts by a transwell cocultured system. Flow cytometry was used to measure CD44 and CD24 expression status on MCF7. ELISA (enzyme-linked immunosorbent assay) was performed to investigate the production of stromal cell-derived factor 1 (SDF-1) in mammosphere cultures subject to various treatments. Mammosphere cells were injected with CAFs and NFs to examine the efficiency of tumorigenity in NOD/SCID mice.

Results

CAFs derived from breast cancer patients were found to be positive for α-smooth muscle actin (α-SMA), exhibiting the traits of myofibroblasts. In addition, CAFs played a central role in promoting the proliferation of CD44⁺CD24^- cells through their ability to secrete SDF-1, which may be mediated to SDF-1/CXCR4 signaling. Moreover, the tumorigenicity of mammosphere cells with CAFs significantly increased as compared to that of mammosphere cells alone or with NFs.

Conclusion

We for the first time investigated the effects of stromal fibroblasts on CD44⁺CD24^- cells and our findings indicated that breast CAFs contribute to CD44⁺CD24^- cell proliferation through the secretion of SDF-1, and which may be important target for therapeutic approaches.     

Dendritic cells loaded with killed breast cancer cells induce differentiation of tumor-specific cytotoxic T lymphocytes

Background

Early clinical trials, mostly in the setting of melanoma, have shown that dendritic cells (DCs) expressing tumor antigens induce some immune responses and some clinical responses. A major difficulty is the extension to other tumors, such as breast carcinoma, for which few defined tumor-associated antigens are available. We have demonstrated, using both prostate carcinoma and melanoma as model systems, that DCs loaded with killed allogeneic tumor cell lines can induce CD8⁺ T cells to differentiate into cytotoxic T lymphocytes (CTLs) specific for shared tumor antigens.

Methods

The present study was designed to determine whether DCs would capture killed breast cancer cells and present their antigens to autologous CD4⁺ and CD8⁺ T cells.

Results

We show that killed breast cancer cells are captured by immature DCs that, after induced maturation, can efficiently present MHC class I and class II peptides to CD8⁺ and CD4⁺ T lymphocytes. The elicited CTLs are able to kill the target cells without a need for pretreatment with interferon gamma. CTLs can be obtained by culturing the DCs loaded with killed breast cancer cells with unseparated peripheral blood lymphocytes, indicating that the DCs can overcome any potential inhibitory effects of breast cancer cells.

Conclusion

Loading DCs with killed breast cancer cells may be considered a novel approach to breast cancer immunotherapy and to identification of shared breast cancer antigens.