RB1 Status in Triple Negative Breast Cancer Cells Dictates Response to Radiation Treatment and Selective Therapeutic Drugs

Triple negative breast cancer (TNBC) includes basal-like and claudin-low subtypes for which only chemotherapy and radiation therapy are currently available. The retinoblastoma (RB1) tumor suppressor is frequently lost in human TNBC. Knockdown of RB1 in luminal BC cells was shown to affect response to endocrine, radiation and several antineoplastic drugs. However, the effect of RB1 status on radiation and chemo-sensitivity in TNBC cells and whether RB1 status affects response to divergent or specific treatment are unknown. Using multiple basal-like and claudin-low cell lines, we hereby demonstrate that RB-negative TNBC cell lines are highly sensitive to gamma-irradiation, and moderately more sensitive to doxorubicin and methotrexate compared to RB-positive TNBC cell lines. In contrast, RB1 status did not affect sensitivity of TNBC cells to multiple other drugs including cisplatin (CDDP), 5-fluorouracil, idarubicin, epirubicin, PRIMA-1^met, fludarabine and PD-0332991, some of which are used to treat TNBC patients. Moreover, a non-biased screen of ∼3400 compounds, including FDA-approved drugs, revealed similar sensitivity of RB-proficient and -deficient TNBC cells. Finally, ESA⁺/CD24^−/low/CD44⁺ cancer stem cells from RB-negative TNBC lines were consistently more sensitive to gamma-irradiation than RB-positive lines, whereas the effect of chemotherapy on the cancer stem cell fraction varied irrespective of RB1 expression. Our results suggest that patients carrying RB-deficient TNBCs would benefit from gamma-irradiation as well as doxorubicin and methotrexate therapy, but not necessarily from many other anti-neoplastic drugs.     

Synergistic anticancer action of quercetin and curcumin against triple-negative breast cancer cell lines

Women with the breast cancer type 1 susceptibility protein (BRCA1) mutation and loss of BRCA1 expression are reported to have an increased risk of triple-negative breast cancer (TNBC). Targeting BRCA1 modulation might offer a therapeutic option to treat TNBC patients. Our studies detected that BRCA1 is poorly expressed in TNBC cell lines and highly expressed in ER⁺ breast cancer cell lines. To modulate BRCA1 expression, we tested two different dietary components to find out if any would induce tumor suppressor genes. We detected that quercetin and curcumin dose-dependently enhanced the BRCA1 expression. Further, a synergistic action of quercetin and curcumin was observed in modulating the BRCA1 level and in inhibiting the cell survival and migration of TNBC cell lines. Quercetin and curcumin appeared to induce BRCA1 promoter histone acetylation. Furthermore, BRCA1 knockdown induced cell survival and cell migration in ER ⁺ cells were significantly decreased by the combined treatment of quercetin and curcumin. Our present study concluded that the combination treatment of quercetin and curcumin acts synergistically to induce anticancer activity against TNBC cells by modulating tumor suppressor genes.     

Salinomycin Promotes Anoikis and Decreases the CD44+/CD24- Stem-Like Population via Inhibition of STAT3 Activation in MDA-MB-231 Cells

Triple-negative breast cancer (TNBC) is an aggressive tumor subtype with an enriched CD44⁺/CD24^- stem-like population. Salinomycin is an antibiotic that has been shown to target cancer stem cells (CSC); however, the mechanisms of action involved have not been well characterized. The objective of the present study was to investigate the effect of salinomycin on cell death, migration, and invasion, as well as CSC-like properties in MDA-MB-231 breast cancer cells. Salinomycin significantly induced anoikis-sensitivity, accompanied by caspase-3 and caspase-8 activation and PARP cleavage, during anchorage-independent growth. Salinomycin treatment also caused a marked suppression of cell migration and invasion with concomitant downregulation of MMP-9 and MMP-2 mRNA levels. Notably, salinomycin inhibited the formation of mammospheres and effectively reduced the CD44⁺/CD24^- stem-like population during anchorage-independent growth. These observations were associated with the inhibition of STAT3 phosphorylation (Tyr705). Furthermore, interleukin-6 (IL-6)-induced STAT3 activation was strongly suppressed by salinomycin challenge. These findings support the notion that salinomycin may be potentially efficacious for targeting breast cancer stem-like cells through the inhibition of STAT3 activation.     

Ovarian cancer cytoreduction induces changes in T cell population subsets reducing immunosuppression

Surgery is the primary therapeutic strategy for most solid tumours; however, modern oncology has established that neoplasms are frequently systemic diseases. Being however a local treatment, the mechanisms through which surgery plays its systemic role remain unknown. We have investigated the influence of cytoreduction on the immune system of primary and recurrent ovarian cancer. All ovarian cancer patients show an increase in CD4⁺CD25⁺FOXP3⁺ circulating cells (CD4 T_reg). CD4/CD8 ratio is increased in primary tumours, but not in recurrent neoplasms. Primary cytoreduction is able to increase circulating CD4 and CD8 effector cells and decrease CD4 naïve T cells. CD4⁺ T_reg cells rapidly decreased after primary tumour debulking, while CD8⁺CD25⁺FOXP3⁺ (CD8 T_reg) cells are not detectable in peripheral blood. Similar results on CD4 T_reg were observed with chemical debulking in women subjected to neoadjuvant chemotherapy. CD4 and CD8 T_reg cells are both present in neoplastic tissue. Interleukin (IL)‐10 serum levels decrease after surgery, while no changes are observed in transforming growth factor‐β₁ and IL‐6 levels. Surgically induced reduction of the immunosuppressive environment results in an increased capacity of CD8⁺ T cells to respond to the recall antigens. None of these changes was observed in patients previously subjected to chemotherapy or affected by recurrent disease. In conclusion, we demonstrate in ovarian cancer that primary debulking is associated with a reduction of circulating T_reg and an increase in CD8 T‐cell function. Debulking plays a beneficial systemic effect by reverting immunosuppression and restoring immunological fitness.     

Putative tumor suppressor cytoglobin promotes aryl hydrocarbon receptor ligand–mediated triple negative breast cancer cell death

Nearly 40 000 women die annually from breast cancer in the United States. Clinically available targeted breast cancer therapy is largely ineffective in triple negative breast cancer (TNBC), characterized by tumors that lack expression of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (Her2). TNBC is associated with a poor prognosis. Previous reports show that aryl hydrocarbon receptor (AhR) partial agonist 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203) selectively inhibits the growth of breast cancer cells, including those of the TNBC subtype. We previously demonstrated that 5F 203 induced the expression of putative tumor suppressor gene cytoglobin (CYGB) in breast cancer cells. In the current study, we determined that 5F 203 induces apoptosis and caspase-3 activation in MDA-MB-468 TNBC cells and in T47D ER⁺ PR ⁺ Her2 ⁻ breast cancer cells. We also show that caspases and CYGB promote 5F 203–mediated apoptosis in MDA-MB-468 cells. 5F 203 induced lysosomal membrane permeabilization (LMP) and cathepsin B release in MDA-MB-468 and T47D cells. In addition, silencing CYGB attenuated the ability of 5F 203 to induce caspase-3/-7 activation, proapoptotic gene expression, LMP, and cathepsin B release in MDA-MB-468 cells. Moreover, 5F 203 induced CYGB protein expression, proapoptotic protein expression, and caspase-3 cleavage in MDA-MB-468 cells and in MDA-MB-468 xenograft tumors grown orthotopically in athymic mice. These data provide a basis for the development of AhR ligands with the potential to restore CYGB expression as a novel strategy to treat TNBC.     

肝癌小鼠外周血及脾脏T淋巴细胞亚群的变化及其意义

目的:观察小鼠原位肝癌模型外周血以及脾脏T淋巴细胞亚群与正常小鼠之间的差异变化,探讨其差异变化的意义。方法:在正常KM小鼠肝脏种植H22细胞,建立小鼠原位模型。采用流式细胞术,以健康正常小鼠为对照,检测肝癌小鼠外周血以及脾脏T淋巴细胞亚群的变化。结果:与健康正常小鼠相比,肝癌小鼠外周血CD4~+T淋巴细胞、CD4~+/CD8~+比例有显著性降低,CD8~+T淋巴细胞显著性升高;脾脏CD3~+、CD4~+T淋巴细胞有显著性降低。结论:小鼠原位肝癌模型外周血以及脾脏T淋巴细胞亚群发生异常,免疫系统紊乱,可以反映小鼠肝癌的发生、发展。     

Inhibition of breast cancer growth via miR-7 suppressing ALDH1A3 activity concomitant with decreasing breast cancer stem cell subpopulation

Breast cancer patients with high expression of aldehyde dehydrogenases (ALDHs) cell population have higher tolerability to chemotherapy since the cells posses a characteristic of breast cancer stem cells (BCSCs) that are resistant to conventional chemotherapy. In this study, we found that the ALDH-positive cells were higher in CD44⁺CD24⁻ and CD44⁺CD24⁻ESA⁺BCSCs than that in both BT549 and MDA-MB-231 cell lines but microRNA-7 (miR-7) level was lower in CD44⁺CD24⁻ and CD44⁺CD24⁻ESA⁺BCSCs than that in MDA-MB-231 cells. Moreover, miR-7 overexpression in MDA-MB-231 cells decreased ALDH1A3 activity by miR-7 directly binding to the 3′-untranslated region of ALDH1A3; while the ALDH1A3 expression was downregulated in MDA-MB-231 cells, the expressions of CD44 and Epithelium Specific Antigen (ESA) were reduced along with decreasing the BCSC subpopulation. Significantly, enforced expression of miR-7 in CD44⁺CD24⁻ESA⁺BCSC markedly inhibited the BCSC-driven xenograft growth in mice by decreasing an expression of ALDH1A3. Collectively, the findings demonstrate the miR-7 inhibits breast cancer growth via suppressing ALDH1A3 activity concomitant with decreasing BCSC subpopulation. This approach may be considered for an investigation on clinical treatment of breast cancers.     

Clinical-scale selection and viral transduction of human naïve and central memory CD8+ T cells for adoptive cell therapy of cancer patients

The adoptive transfer of lymphocytes genetically engineered to express tumor-specific antigen receptors is a potent strategy to treat cancer patients. T lymphocyte subsets, such as naïve or central memory T cells, selected in vitro prior to genetic engineering have been extensively investigated in preclinical mouse models, where they demonstrated improved therapeutic efficacy. However, so far, this is challenging to realize in the clinical setting, since good manufacturing practices (GMP) procedures for complex cell sorting and genetic manipulation are limited. To be able to directly compare the immunological attributes and therapeutic efficacy of naïve (T_N) and central memory (T_CM) CD8⁺ T cells, we investigated clinical-scale procedures for their parallel selection and in vitro manipulation. We also evaluated currently available GMP-grade reagents for stimulation of T cell subsets, including a new type of anti-CD3/anti-CD28 nanomatrix. An optimized protocol was established for the isolation of both CD8⁺ T_N cells (CD4^?CD62L⁺CD45RA⁺) and CD8⁺ T_CM (CD4^?CD62L⁺CD45RA^?) from a single patient. The highly enriched T cell subsets can be efficiently transduced and expanded to large cell numbers, sufficient for clinical applications and equivalent to or better than current cell and gene therapy approaches with unselected lymphocyte populations. The GMP protocols for selection of T_N and T_CM we reported here will be the basis for clinical trials analyzing safety, in vivo persistence and clinical efficacy in cancer patients and will help to generate a more reliable and efficacious cellular product.     

Midostaurin preferentially attenuates proliferation of triple-negative breast cancer cell lines through inhibition of Aurora kinase family

Background

Breast cancer is classified into three subtypes by the expression of biomarker receptors such as hormone receptors and human epidermal growth factor receptor 2. Triple-negative breast cancer (TNBC) expresses none of these receptors and has an aggressive phenotype with a poor prognosis, which is insensitive to the drugs that target the hormone receptors and human epidermal growth factor receptor 2. It is, thus, required to develop an effective therapeutic reagent to treat TNBC.

Results

The study using a panel of 19 breast cancer cell lines revealed that midostaurin, a multi-target protein kinase inhibitor, suppresses preferentially the growth of TNBC cells comparing with non-TNBC cells. Clustering analysis of the drug activity data for the panel of cancer cell lines predicted that midostaurin shares the target with Aurora kinase inhibitors. Following studies indicated that midostaurin attenuates the phosphorylation reaction mediated by Aurora kinase in the cells and directly inhibits this protein kinase in vitro, and that this reagent induces apoptosis accompanying accumulation of 4N and 8N DNA cells in TNBC cells.

Conclusion

Midostaurin suppresses the proliferation of TNBC cells among the breast cancer cell lines presumably through the inhibition of the Aurora kinase family. The precise study of midostaurin on cell growth will contribute to the development of the drug for the treatment of TNBC.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0150-2) contains supplementary material, which is available to authorized users.     

Grain-sized moxibustion promotes NK cell antitumour immunity by inhibiting adrenergic signalling in non–small cell lung cancer

Lung cancer is the leading cause of cancer-related death worldwide, and non–small cell lung cancer (NSCLC) accounts for 85% of lung cancer diagnoses. As an ancient therapy, moxibustion has been used to treat cancer-related symptoms in clinical practice. However, its antitumour effect on NSCLC remains largely unexplored. In the present study, a Lewis lung cancer (LLC) xenograft tumour model was established, and grain-sized moxibustion (gMoxi) was performed at the acupoint of Zusanli (ST36). Flow cytometry and RNA sequencing (RNA-Seq) were used to access the immune cell phenotype, cytotoxicity and gene expression. PK136, propranolol and epinephrine were used for natural killer (NK) cell depletion, β-adrenoceptor blockade and activation, respectively. Results showed that gMoxi significantly inhibited LLC tumour growth. Moreover, gMoxi significantly increased the proportion, infiltration and activation of NK cells, whereas it did not affect CD4⁺ and CD8⁺ T cells. NK cell depletion reversed gMoxi-mediated tumour regression. LLC tumour RNA-Seq indicated that these effects might be related to the inhibition of adrenergic signalling. Surely, β-blocker propranolol clearly inhibited LLC tumour growth and promoted NK cells, and gMoxi no longer increased tumour regression and promoted NK cells after propranolol treatment. Epinephrine could inhibit NK cell activity, and gMoxi significantly inhibited tumour growth and promoted NK cells after epinephrine treatment. These results demonstrated that gMoxi could promote NK cell antitumour immunity by inhibiting adrenergic signalling, suggesting that gMoxi could be used as a promising therapeutic regimen for the treatment of NSCLC, and it had a great potential in NK cell–based cancer immunotherapy.     

A preliminary study of the role of extracellular -5′- nucleotidase in breast cancer stem cells and epithelial-mesenchymal transition

Tumor stem cell theory may well explain a variety of malignant behaviors of tumors. Cells undergoing epithelial-mesenchymal transition (EMT) share many characteristics with tumor stem cells. Our previous studies showed that extracellular -5'- nucleotidase (CD73), one of the important surface markers of mesenchymal stem cells, may promote growth and metastasis of breast cancer cells both in vivo and in vitro. In this study, we assessed breast cancer stem cell (BCSC) markers [acetaldehyde dehydrogenase (ALDH)⁺ and CD44⁺CD24^?] in various breast cancer cell lines with flow cytometry after overexpression (by lentivirus infection) or suppression (by siRNA interference) of CD73. We measured CD73 expression in breast cancer mammospheres with real-time PCR and western blots. Finally, we examined the expression of CD73 and EMT markers in different breast cancer cell lines, as well as in mammary cells (MCF10A) that underwent EMT induced by transforming growth factor beta (TGF-β). We found that CD73 positively correlated with ALDH⁺ or CD44⁺CD24^? subsets of breast cancer cells. CD73 was expressed more in breast cancer mammospheres than in adherent cells. CD73 and mesenchymal marker expression was higher in breast cancer cells with more malignant features, while CD73 was lower in low malignant breast cancer cells with higher epithelial markers. Furthermore, CD73 expression increased during the process of TGF-β-induced EMT. Our results indicate that CD73 may play an important role in BCSCs.     

LIPH promotes metastasis by enriching stem‐like cells in triple‐negative breast cancer

Lipase member H (LIPH), a novel member of the triglyceride lipase family. The clinical implications of its expression in breast cancer are still unclear. Therefore, in this study, we investigated the associations between LIPH and the tumorigenic behaviours of 144 triple‐negative breast cancer (TNBC) patients. The ratio and mammosphere‐forming ability of CD44+/CD24? stem‐like cells were tested. The role of LIPH in breast cancer cell migration and invasion was also evaluated. In addition, the effect of LIPH silencing on mitochondrial respiration was determined using the Seahorse assay. Finally, the effect of LIPH silencing on protein expression was determined via tandem mass tag‐based spectrometry and Western blotting. We found that LIPH expression was associated with metastasis in lymph nodes and distant organs (P = 0.025), resulting in poor survival among breast cancer patients (P = 0.027). LIPH knockdown significantly decreased both the ratio of CD44⁺/CD24^? stem‐like cells and their mammosphere‐forming ability. LIPH silencing promoted apoptosis, arrested cell cycle in the G2/M phase, mitigated the oxidation‐related oxygen consumption rate in the mitochondria, and reduced metabolism. LIPH inhibited adhesion between tumour cells and enhanced the epithelial‐mesenchymal transition. Tandem mass spectrometric analysis presented 68 proteins were differentially expressed in LIPH‐silenced cells and LIPH‐mediated modulation of tumour cell adhesion depended on integrin‐related CAPN2 and paxillin signalling. Overall, our findings provided strong evidence that LIPH up‐regulation promoted metastasis and the stemness of TNBC cells. Therefore, targeting LIPH is a potentially viable strategy for preventing metastasis in TNBC.     

The presence of LC3B puncta and HMGB1 expression in malignant cells correlate with the immune infiltrate in breast cancer

Several cell-intrinsic alterations have poor prognostic features in human breast cancer, as exemplified by the absence of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 β)-positive puncta in the cytoplasm (which indicates reduced autophagic flux) or the loss of nuclear HMGB1 expression by malignant cells. It is well established that breast cancer is under strong immunosurveillance, as reflected by the fact that scarce infiltration of the malignant lesion by CD8⁺ cytotoxic T lymphocytes or comparatively dense infiltration by immunosuppressive cell types (such as FOXP3⁺ regulatory T cells or CD68⁺ tumor-associated macrophages), resulting in low CD8⁺:FOXP3⁺ or CD8⁺:CD68⁺ ratios, has a negative prognostic impact. Here, we reveal the surprising finding that cell-intrinsic features may influence the composition of the immune infiltrate in human breast cancer. Thus, the absence of LC3B puncta is correlated with intratumoral (but not peritumoral) infiltration by fewer CD8⁺ cells and more FOXP3⁺ or CD68⁺ cells, resulting in a major drop in the CD8⁺:FOXP3⁺ or CD8⁺:CD68⁺ ratios. Moreover, absence of HMGB1 expression in nuclei correlated with a general drop in all immune effectors, in particular FOXP3⁺ and CD68⁺ cells, both within the tumor and close to it. Combined analysis of LC3B puncta and HMGB1 expression allowed for improved stratification of patients with respect to the characteristics of their immune infiltrate as well as overall and metastasis-free survival. It can be speculated that blocked autophagy in, or HMGB1 loss from, cancer cells may favor tumor progression due to their negative impact on anticancer immunosurveillance.     

Interleukin 2 and interleukin 10 function synergistically to promote CD8+ T cell cytotoxicity,which is suppressed by regulatory T cells in breast cancer

The precise role of interleukin (IL)-10 in breast cancer is not clear. Previous studies suggested a tumor-promoting role of IL-10 in breast cancer, whereas recent discoveries that IL-10 activated and expanded tumor-resident CD8⁺ T cells challenged the traditional view. Here, we investigated the role of IL-10 in HLA-A2-positive breast cancer patients with Grade III, Stage IIA or IIB in-situ and invasive ductal carcinoma, and compared it with that of IL-2, the canonical CD8⁺ T cell growth factor. We first observed that breast cancer patients presented higher serum levels of IL-2 and IL-10 than healthy controls. Upon prolonged TCR stimulation, peripheral blood CD8⁺ T cells from breast cancer patients tended to undergo apoptosis, which could be prevented by the addition of IL-2 and/or IL-10. The cytotoxicity of TCR-activated CD8⁺ T cells was also enhanced by exogenous IL-2 and/or IL-10. Interestingly, IL-2 and IL-10 demonstrated synergistic effects, since the enhancement in CD8⁺ T cell function when both cytokines were added was greater than the sum of the improvements mediated by each individual cytokine. IL-10 by itself could not promote the proliferation of CD8⁺ T cells but could significantly enhance IL-2-mediated promotion of CD8⁺ T cell proliferation. In addition, the cytotoxicity of tumor-infiltrating CD8⁺ T cells in breast tumor was elevated when both IL-2 and IL-10 were present but not when either one was absent. This synergistic effect was stopped by CD4⁺CD25⁺ regulatory T cells (Treg), which depleted IL-2 in a cell number-dependent manner. Together, these results demonstrated that IL-2 and IL-10 could work synergistically to improve the survival, proliferation, and cytotoxicity of activated CD8⁺ T cells, an effect suppressible by CD4⁺CD25⁺ Treg cells.     

Proangiogenic TIE2+/CD31+ macrophages are the predominant population of tumor-associated macrophages infiltrating metastatic lymph nodes

Tumor-associated macrophages (TAMs) accumulate in various cancers and promote tumor angiogenesis and metastasis, and thus may be ideal targets for the clinical diagnosis of tumor metastasis with high specificity. However, there are few specific markers to distinguish between TAMs and normal or inflammatory macrophages. Here, we show that TAMs localize in green fluorescent protein-labeled tumors of metastatic lymph nodes (MLNs) from B16F1 melanoma cells but not in necrotic tumor regions, suggesting that TAMs may promote the growth of tumor cells and the progression of tumor metastasis. Furthermore, we isolated pure populations of TAMs from MLNs and characterized their gene expression signatures compared to peritoneal macrophages (PMs), and found that TAMs significantly overexpress immunosuppressive cytokines such as IL-4, IL-10, and TGF-β as well as proangiogenic factors such as VEGF, TIE2, and CD31. Notably, immunological analysis revealed that TIE2⁺/CD31⁺ macrophages constitute the predominant population of TAMs that infiltrate MLNs, distinct from tissue or inflammatory macrophages. Importantly, these TIE2⁺/CD31⁺ macrophages also heavily infiltrated MLNs from human breast cancer biopsies but not reactive hyperplastic LNs. Thus, TIE2⁺/CD31⁺ macrophages may be a unique histopathological biomarker for detecting metastasis in clinical diagnosis, and a novel and promising target for TAM-specific cancer therapy.     

肺癌患者外周血淋巴细胞亚群水平的表达及临床意义

目的:探讨肺癌患者外周血淋巴细胞亚群水平的表达及临床意义。方法:选择2016年3月~2017年3月期间我院收治的88例肺癌患者作为研究组,选择同期于我院进行健康体检的88例受检者作为对照组。两组研究对象均通过流式细胞仪检测外周血T淋巴细胞亚群水平。观察对比两组研究对象外周血T淋巴细胞亚群的表达水平,以及研究组不同临床病理特征患者外周血T淋巴细胞亚群的表达水平。结果:研究组CD4~+/CD8~+、CD4~+、CD3~+表达水平均低于对照组,CD8~+表达水平高于对照组,差异具有统计学意义(P0.05)。研究组Ⅲ期与Ⅳ期患者CD4~+/CD8~+、CD4~+、CD3~+表达水平均低于Ⅰ期与Ⅱ期,Ⅲ期与Ⅳ期患者CD8~+表达水平高于Ⅰ期与Ⅱ期(P0.05);小细胞肺癌患者CD4~+/CD8~+、CD4~+、CD3~+表达水平均低于非小细胞肺癌患者,CD8~+表达水平高于非小细胞肺癌患者,差异具有统计学意义(P0.05)。不同性别、年龄、肿瘤分化程度肺癌患者CD4~+/CD8~+、CD8~+、CD4~+、CD3~+水平无统计学差异(P0.05)。结论:肺癌患者外周血淋巴细胞亚群水平的表达呈现异常状态,且表达水平与疾病的分期和病理分型有关。