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Breast cancer stands as a formidable global health challenge for women. While neoantigens exhibit efficacy in activating T cells specific to cancer and instigating anti-tumor immune responses, the accuracy of neoantigen prediction remains suboptimal. In this study, we identified neoantigens from the patient-derived breast cancer cells, PC-B-142CA and PC-B-148CA cells, utilizing whole-genome and RNA sequencing. The pVAC-Seq pipeline was employed, with minor modification incorporating criteria (1) binding affinity of mutant (MT) peptide with HLA (IC50 MT) ≤ 500 nm in 3 of 5 algorithms and (2) IC50 wild type (WT)/MT > 1. Sequencing results unveiled 2513 and 3490 somatic mutations, and 646 and 652 non-synonymous mutations in PC-B-142CA and PC-B-148CA, respectively. We selected the top 3 neoantigens to perform molecular dynamic simulation and synthesized 9-12 amino acid neoantigen peptides, which were then pulsed onto healthy donor peripheral blood mononuclear cells (PBMCs). Results demonstrated that T cells activated by ADGRL1E274K, PARP1E619K, and SEC14L2R43Q peptides identified from PC-B-142CA exhibited significantly increased production of interferon-gamma (IFN-γ), while PARP1E619K and SEC14L2R43Q peptides induced the expression of CD107a on T cells. The % tumor cell lysis was notably enhanced by T cells activated with MT peptides across all three healthy donors. Moreover, ALKBH6V83M and GAAI823T peptides from PC-B-148CA remarkably stimulated IFN-γ- and CD107a-positive T cells, displaying high cell-killing activity against target cancer cells. In summary, our findings underscore the successful identification of neoantigens with anti-tumor T cell functions and highlight the potential of personalized neoantigens as a promising avenue for breast cancer treatment.
Assuntos
Neoplasias da Mama , Feminino , Humanos , Leucócitos Mononucleares , Linfócitos T , Algoritmos , AnticorposRESUMO
BACKGROUND: Carcinoma-associated fibroblasts (CAFs) play a critical role in cancer progression and immune cell modulation. In this study, it was aimed to evaluate the roles of CAFs-derived IL-6 in doxorubicin (Dox) resistance and PD-L1-mediated chimeric antigenic receptor (CAR)-T cell resistance in breast cancer (BCA). METHODS: CAF conditioned-media (CM) were collected, and the IL-6 level was measured by ELISA. CAF-CM were treated in MDA-MB-231 and HCC70 TNBC cell lines and siIL-6 receptor (IL-6R) knocked down (KD) cells to determine the effect of CAF-derived IL-6 on Dox resistance by flow cytometry and on increased PD-L1 through STAT3, AKT and ERK1/2 pathways by Western blot analysis. After pre-treating with CM, the folate receptor alpha (FRα)-CAR T cell cytotoxicity was evaluated in 2D and 3D spheroid culture assays. RESULTS: The results showed a significant level of IL-6 in CAF-CM compared to that of normal fibroblasts (NFs). The CM with high IL-6 level significantly induced Dox resistance; and PD-L1 expression through STAT3 and AKT pathways in MDA-MB-231 and HCC70 cells. These induction effects were attenuated in siIL-6R KD cells. Moreover, the TNBC cell lines that were CM-treated with STAT3 and an AKT inhibitor had a reduced effect of IL-6 on PD-L1 expression. BCA cells with high IL-6 containing-CM treatment had resistance to cancer cell killing by FRα CAR-T cells compared to untreated cells. CONCLUSION: These results highlight CAF-derived IL-6 in the resistance of chemotherapy and T cell therapy. Using inhibitors of IL6-STAT3/AKT-PD-L1 axis may provide a potential benefit of Dox and CAR-T cell therapies in BCA patients.
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Fibroblastos Associados a Câncer , Receptores de Antígenos Quiméricos , Neoplasias de Mama Triplo Negativas , Humanos , Interleucina-6/genética , Proteínas Proto-Oncogênicas c-akt , Antígeno B7-H1/genética , Neoplasias de Mama Triplo Negativas/genética , Linfócitos T , Fator de Transcrição STAT3/genéticaRESUMO
BACKGROUND: High-mobility group box 1 (HMGB1) is increased in breast cancer cells as the result of exposure to the secreted substances from cancer-associated fibroblasts and plays a crucial role in cancer progression and drug resistance. Its effect, however, on the expression of programmed death ligand 1 (PD-L1) in breast cancer cells has not been investigated. This study aimed to investigate the mechanism of HMGB1 through receptors for advanced glycation end products (RAGE) on cell migration/invasion and PD-L1 expression in breast cancer cells. METHODS: A 3-dimensional (3-D) migration and invasion assay and Western blotting analysis to evaluate the function and the mechanism under recombinant HMGB1 (rHMGB1) treatment with knockdown of RAGE using shRAGE and PI3K/AKT inhibitors was performed. RESULTS: The results revealed that rHMGB1 induced MDA-MB-231 cell migration and invasion. The knockdown of RAGE using shRAGE and PI3K/AKT inhibitors attenuated 3-D migration and invasion in response to rHMGB1 compared to mock cells. PD-L1 up-regulation was observed in both parental MDA-MB-231 (P) and MDA-MB-231 metastasis to bone marrow (BM) cells treated with rHMGB1, and these effects were alleviated in RAGE-knock down (KD) breast cancer cells as well as in PI3K/AKT inhibitor-treated cells. CONCLUSIONS: Collectively, these findings indicate that HMGB1-RAGE through PI3K/AKT signaling promotes not only breast cancer cell invasion but also PD-L1 expression which leads to the destruction of the effector T cells. The attenuating HMGB1-RAGE-PI3K/AKT pathway may help to attenuate breast cancer cell aggressive phenotypes.
Assuntos
Antígenos de Neoplasias , Antígeno B7-H1 , Neoplasias da Mama , Proteína HMGB1 , Proteínas Quinases Ativadas por Mitógeno , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Antígenos de Neoplasias/metabolismo , Antígeno B7-H1/biossíntese , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Feminino , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Invasividade Neoplásica , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor para Produtos Finais de Glicação Avançada , Transdução de SinaisRESUMO
BACKGROUND: Chemoresistance is one of the main problems in treatment of cancer. Periostin (PN) is a stromal protein which is mostly secreted from cancer associated fibroblasts in the tumor microenvironment and can promote cancer progression including cell survival, metastasis, and chemoresistance. The main objective of this study was to develop an anti-PN peptide from the bacteriophage library to overcome PN effects in breast cancer (BCA) cells. METHODS: A twelve amino acids bacteriophage display library was used for biopanning against the PN active site. A selected clone was sequenced and analyzed for peptide primary structure. A peptide was synthesized and tested for the binding affinity to PN. PN effects including a proliferation, migration and a drug sensitivity test were performed using PN overexpression BCA cells or PN treatment and inhibited by an anti-PN peptide. An intracellular signaling mechanism of inhibition was studied by western blot analysis. Lastly, PN expressions in BCA patients were analyzed along with clinical data. RESULTS: The results showed that a candidate anti-PN peptide was synthesized and showed affinity binding to PN. PN could increase proliferation and migration of BCA cells and these effects could be inhibited by an anti-PN peptide. There was significant resistance to doxorubicin in PN-overexpressed BCA cells and this effect could be reversed by an anti-PN peptide in associations with phosphorylation of AKT and expression of survivin. In BCA patients, serum PN showed a correlation with tissue PN expression but there was no significant correlation with clinical data. CONCLUSIONS: This finding supports that anti-PN peptide is expected to be used in the development of peptide therapy to reduce PN-induced chemoresistance in BCA.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Moléculas de Adesão Celular/antagonistas & inibidores , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Engenharia de Proteínas , Antibióticos Antineoplásicos/farmacologia , Apoptose , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Fibroblastos Associados a Câncer/efeitos dos fármacos , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Proliferação de Células , Feminino , Humanos , Fragmentos de Peptídeos/química , Prognóstico , Células Tumorais Cultivadas , Microambiente TumoralRESUMO
Colorectal cancer (CRC) is one of the most fatal cancers with highly invasive properties. The progression of CRC is determined by the driving force of periostin (PN) from cancer-associated fibroblasts (CAFs) in the tumour microenvironment. This present work aims to investigate autophagy-mediated CRC invasion via the receptor integrin (ITG) by PN. The level of PN in 410 clinical CRC tissues was found increased and was an independent poor prognosis marker (HR = 2.578, 95% CI = 1.218-5.457, P-value = .013) with a significant correlation with overall survival time (P-value < .001). PN activated proliferation, migration and invasion of CRC cells, but with reduced autophagy. Interestingly, the reduction of LC3 autophagic protein corresponded to the increased ability of CRC cell migration. The siITGα5-treated HT-29 and siITGß4-treated HCT-116 CRC cells attenuated epithelial-to-mesenchymal transitions (EMT)-related genes and pAKT compared with those in siITG-untreated cells. The reduction of pAKT by a PI3K inhibitor significantly restored autophagy in CRC cells. These evidences confirmed the effect of PN through either ITGα5ß1 or ITGα6ß4 and the AKT-dependent pathway to control autophagy-regulated cell migration. In conclusion, these results exhibited the impact of PN activation of ITGα5ß1 or ITGα6ß4 through pAKT in autophagy-mediated EMT and migration in CRC cells.
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Moléculas de Adesão Celular/metabolismo , Neoplasias Colorretais/metabolismo , Integrina alfa6/metabolismo , Integrina beta1/metabolismo , Integrina beta4/metabolismo , Integrinas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Idoso , Autofagia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Fosfatidilinositol 3-Quinases/metabolismo , Prognóstico , Modelos de Riscos Proporcionais , Inibidores de Proteínas Quinases/farmacologia , Resultado do TratamentoRESUMO
Cholangiocarcinoma (CCA) is a very aggressive cancer arising from the malignant transformation of cholangiocytes. Intrahepatic CCA is associated with reactive inflammation and intense fibrosis of the hepatobiliary tract. Dihydroartemisinin (DHA), the active compound found in Artemisia annua, has been shown to possess anti-tumor activity in a variety of human cancers, including hepatoma. Here, we tested the ability of DHA to specifically kill CCA cells and have investigated the underlying mechanisms. DHA induced both apoptosis and autophagy-dependent caspase-independent cell death in many CCA cell lines, while being slightly toxic to immortalized cholangiocytes. DHA induced the expression of many apoptosis- and autophagy-related genes in CCA cells. In particular, it greatly induced the expression of DAPK1, and reduced the interaction of BECLIN1 with BCL-2 while promoting its interaction with PI3KC3. Genetic silencing of DAPK1 prevented DHA-induced autophagy. Pharmacologic and genetic inhibition of BECLIN1 function prevented autophagy and cell death induced by DHA in CCA cells. These data unravel a novel pathway of DHA cancer toxicity and open the possibility to introduce DHA in the therapeutic regimen for the treatment of CCA.
Assuntos
Artemisininas/farmacologia , Neoplasias dos Ductos Biliares/metabolismo , Colangiocarcinoma/metabolismo , Transdução de Sinais/efeitos dos fármacos , Artemisia annua/química , Autofagia , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Neoplasias dos Ductos Biliares/tratamento farmacológico , Neoplasias dos Ductos Biliares/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/genética , Proteínas Quinases Associadas com Morte Celular/genética , Proteínas Quinases Associadas com Morte Celular/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Resveratrol, a natural polyphenol present in a variety of food stuff, has been shown to exert preventive and curative anticancer activity in several in vitro and in vivo models. Such chemopreventive/anticancer activity has been linked to biochemical and epigenetic modifications of multiple pathways involved in carcinogenesis and metastasization. In this commentary, we focus on the recent work done in our laboratory showing that resveratrol has potential to prevent and cure cancer by promoting epigenetic-mediated autophagy-dependent tumor dormancy, an effect associated with re-education of the cancer-associated fibroblasts and reduced production of inflammatory cytokines in the tumor microenvironment. The clinical translation of the current knowledge on resveratrol anticancer activity is also discussed.
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Colorectal cancer (CRC) is the third most common malignancy cause of cancer-related mortality worldwide. Epithelial-mesenchymal transition (EMT) promotes cancer metastasis and a tumour-based Glasgow EMT score was associated with adverse clinical features and poor prognosis. In this study, the impact of using the established five tumour-based EMT markers consisting of E-cadherin (E-cad), ß-catenin (ß-cat), Snail, Zeb-1, and Fascin in combination with the stromal periostin (PN) on the prediction of CRC patients' prognosis were invesigated. Formalin-fixed paraffin-embedded tissues of 202 CRC patients were studies the expressions of E-cad, ß-cat, Snail, Zeb-1, Fascin, and PN by immunohistochemistry. Individually, cytoplasmic Fascin (Fc), cytoplasmic Snail (Sc), nuclear Snail (Sn), stromal Snail (Ss), and stromal PN (Ps) were significantly associated with reduced survival. A combination of Ps with Fc, Fs, and Sn was observed in 2 patterns including combined Fc, Fs, and Ps (FcFsPs) and Fc, Sn, and Ps (FcSnPs). These combinations enhanced the prognostic power compared to individual EMT markers and were independent prognostic markers. As the previously established scoring method required five markers and stringent criteria, its clinical use might be limited. Therefore, using these novel combined prognostic markers, either FcFsPs or FcSnPs, may be useful in predicting CRC patient outcomes.
Assuntos
Biomarcadores Tumorais , Proteínas de Transporte , Moléculas de Adesão Celular , Neoplasias Colorretais , Transição Epitelial-Mesenquimal , Proteínas dos Microfilamentos , Fatores de Transcrição da Família Snail , Humanos , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Neoplasias Colorretais/mortalidade , Fatores de Transcrição da Família Snail/metabolismo , Moléculas de Adesão Celular/metabolismo , Prognóstico , Feminino , Masculino , Pessoa de Meia-Idade , Proteínas de Transporte/metabolismo , Proteínas dos Microfilamentos/metabolismo , Idoso , Biomarcadores Tumorais/metabolismo , Adulto , Caderinas/metabolismo , Fatores de Transcrição/metabolismo , beta Catenina/metabolismo , Idoso de 80 Anos ou mais , PeriostinaRESUMO
Cholangiocarcinoma (CCA), the malignant tumor of bile duct epithelial cells, is a relatively rare yet highly lethal cancer. In this work, we tested the ability of Resveratrol (RV) to prevent and cure CCA xenograft in nude mice and investigated molecular mechanisms underpinning such anticancer effect. Human CCA cells were xenografted in mice that were or not treated prior to or after to transplantation with RV. Tumor growth was monitored and analyzed for the markers of cell proliferation, apoptosis, and autophagy. TCGA was interrogated for the molecules possibly targeted by RV. RV could inhibit the growth of human CCA xenograft when administered after implantation and could reduce the growth or even impair the implantation of the tumors when administered prior the transplantation. RV inhibited CCA cell proliferation, induced apoptosis with autophagy, and strongly reduced the presence of CAFs and production of IL-6. Interrogation of CCA dataset in TCGA database revealed that the expression of IL-6 Receptor (IL-6R) inversely correlated with that of MAP-LC3 and BECLIN-1, and that low expression of IL-6R and of MIK67, two pathways downregulated by RV, associated with better survival of CCA patients. Our data demonstrate that RV elicits a strong preventive and curative anticancer effect in CCA by limiting the formation of CAFs and their release of IL-6, and this results in up-regulation of autophagy and apoptosis in the cancer cells. These findings support the clinical use of RV as a primary line of prevention in patients exposed at risk and as an adjuvant therapeutics in CCA patients.
Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Humanos , Animais , Camundongos , Resveratrol/farmacologia , Resveratrol/uso terapêutico , Xenoenxertos , Interleucina-6/genética , Camundongos Nus , Neoplasias dos Ductos Biliares/tratamento farmacológico , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/prevenção & controle , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/genética , Colangiocarcinoma/prevenção & controle , Proliferação de Células , Ductos Biliares Intra-Hepáticos/patologia , Linhagem Celular Tumoral , ApoptoseRESUMO
The benefits of treating several types of cancers using immunotherapy have recently been established. The overexpression of nucleolin (NCL) in a number of types of cancer provides an attractive antigen target for the development of novel anticancer immunotherapeutic treatments. NCL is a multifunctional protein abundantly distributed in the nucleus, cytoplasm and cell membrane. It influences carcinogenesis, and the proliferation, survival and metastasis of cancer cells, leading to cancer progression. Additionally, the metaanalysis of total and cytoplasmic NCL overexpression indicates a poor prognosis of patients with breast cancer. The AS1411 aptamers currently appear to have therapeutic action in the phase II clinical trial. The authors' research group has recently explored the anticancer function of NCL through the activation of T cells by dendritic cellbased immunotherapy. The present review describes and discusses the mechanisms through which the multiple functions of NCL can participate in the progression of cancer. In addition, the studies that define the utility of NCLdependent anticancer therapies are summarized, with specific focus being paid to cancer immunotherapeutic approaches.
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Neoplasias da Mama , Fosfoproteínas , Humanos , Feminino , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Imunoterapia , NucleolinaRESUMO
Tenosynovial giant cell tumor (TGCT) is a mesenchymal tumor derived from the synovium of the tendon sheath and joints, most frequently in the large joints. The standard of care for TGCTs is surgical resection. A new targeting approach for treating TGCTs has emerged from studies on the role of the CSF1/CSF1 receptor (CSF1R) in controlling cell survival and proliferation during the pathogenesis of TGCTs. We established four novel cell lines isolated from the primary tumor tissues of patients with TGCTs. The cell lines were designated Si-TGCT-1, Si-TGCT-2, Si-TGCT-3, and Si-TGCT-4, and the TGCT cells were characterized by CSF1R and CD68. These TGCT cells were then checked for cell proliferation using an MTT assay and three-dimensional spheroid. The responses to pexidartinib (PLX3397) and sotuletinib (BLZ945) were evaluated by two-dimensional MTT assays. All cells were positive for αsmooth muscle actin (αSMA), fibroblast activation protein (FAP), CSF1R, and CD68. Except for Si-TGCT-4, all TGCT cells had high CSF1R expressions. The cells exhibited continuous growth as three-dimensional spheroids formed. Treatment with pexidartinib and sotuletinib inhibited TGCT cell growth and induced cell apoptosis correlated with the CSF1R level. Only Si-TGCT-4 cells demonstrated resistance to the drugs. In addition, the BAX/BCL-2 ratio increased in cells treated with pexidartinib and sotuletinib. With the four novel TGCT cell lines, we have an excellent model for further in vitro and in vivo studies.
Assuntos
Tumor de Células Gigantes de Bainha Tendinosa , Receptor de Fator Estimulador de Colônias de Macrófagos , Humanos , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Tumor de Células Gigantes de Bainha Tendinosa/tratamento farmacológico , Tumor de Células Gigantes de Bainha Tendinosa/genética , Linhagem CelularRESUMO
Nucleolin (NCL) is a multifunctional protein expressed in the nucleus, cytoplasm, and cell membrane. Overexpression of NCL has a controversial role as a poor prognostic marker in cancers. In this study, a meta-analysis was performed to evaluate the prognostic value of NCL in different subcellular localizations (cytoplasmic (CyNCL) and nuclear (NuNCL)) across a range of cancers. PubMed was searched for relevant publications. Data were extracted and analyzed from 12 studies involving 1221 patients with eight cancer types. The results revealed high total NCL was significantly associated with poor overall survival (OS) (HR = 2.85 (1.94, 4.91), p < 0.00001, I2 = 59%) and short disease-free survival (DFS) (HR = 3.57 (2.76, 4.62), p < 0.00001, I2 = 2%). High CyNCL was significantly associated with poor OS (HR = 4.32 (3.01, 6.19), p < 0.00001, I2 = 0%) and short DFS (HR = 3.00 (2.17, 4.15), p < 0.00001, I2 = 0%). In contrast, high NuNCL correlated with increased patient OS (HR = 0.42 (0.20, 0.86), p = 0.02, I2 = 66%), with no significant correlation to DFS observed (HR = 0.46 (0.19, 1.14), p = 0.09, I2 = 57%). This study supports the role of subcellular NCL as a poor prognostic cancer biomarker.
Assuntos
Neoplasias , Fosfoproteínas , Intervalo Livre de Doença , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/genética , NucleolinaRESUMO
Triple negative breast cancer (TNBC) lacks targeted treatment resulting in poor prognosis. Targeting overexpressing mesothelin (MSLN) using MSLNspecific T cells is an attractive treatment approach and the aim of the present study. The expression of MSLN in human TNBC paraffin sections was analyzed by immunohistochemistry. Lentiviral vector harbored granulocytemacrophage colony stimulating factor (GMCSF), interleukin4 (IL4) and MSLN cDNAs was constructed to generate selfdifferentiated myeloidderived antigenpresentingcells reactive against tumor expressing MSLN dendritic cell (MSLNSmartDC) for MSLNspecific T cell activation. The results showed high MSLN in 32.8% of all breast cancer subtypes and 57% in TNBC. High MSLN was significantly associated with TNBC subtype and the absence of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2. MSLNSmartDC exhibited comparable phenotype to DC generated by exogenous cytokine treatment and an addition of 40s ribosomal protein subunit 3 (RPS3), a tolllike receptor 4 ligand, enhanced DC maturation and function by upregulation of CD40, CD80 and CD83 expressions and IL12p70 secretion. MSLNspecific CD8+CD69+ IFNγ+ T cells were detected in T cells activated by both MSLNSmartDC and RPS3MSLNSmartDC. MSLNspecific T cells activated by these DCs showed more specific killing capability against naturally expressed MSLNHCC70 and artificially MSLNoverexpressing MDAMB231 compared with parental MDAMB231 in both two dimensional (2D) and 3Dculture systems. In conclusion, the results demonstrated the efficacy of MSLNSmartDC to promote MSLNspecific T cells response against TNBC and RPS3 can enhance the cytolytic activity of these T cells providing an alternative treatment approach for patients with TNBC.
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Células Dendríticas , Mesotelina , Proteínas Ribossômicas , Linfócitos T , Neoplasias de Mama Triplo Negativas , Linhagem Celular Tumoral , Células Dendríticas/metabolismo , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Humanos , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Linfócitos T/imunologia , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Dendritic cell (DC)-based T-cell activation is an alternative immunotherapy in breast cancer. The anti-programmed death ligand 1 (PD-L1) can enhance T-cell function. Nucleolin (NCL) is overexpressed in triple-negative breast cancer (TNBC). The regulation of PD-L1 expression through autophagy and the anti-PD-L1 peptide to help sensitize T cells for NCL-positive TNBC cell killing has not been evaluated. Results showed the worst clinical outcome in patients with high NCL and PD-L1. Self-differentiated myeloid-derived antigen-presenting cells reactive against tumors presenting NCL or SmartDCs-NCL producing GM-CSF and IL-4, could activate NCL-specific T cells. SmartDCs-NCL plus recombinant human ribosomal protein substrate 3 (RPS3) successfully induced maturation and activation of DCs characterized by the reduction of CD14 and the induction of CD11c, CD40, CD80, CD83, CD86, and HLA-DR. Interestingly, SmartDCs-NCL plus RPS3 in combination with anti-PD-L1 peptide revealed significant killing activity of the effector NCL-specific T cells against NCLHigh/PD-L1High MDA-MB-231 and NCLHigh/PD-L1High HCC70 TNBC cells at the effector: a target ratio of 5:1 in 2-D and 10:1 in the 3-D culture system; and increments of IFNγ by the ELISpot assay. No killing effect was revealed in MCF-10A normal mammary cells. Mechanistically, NCL-specific T-cell-mediated TNBC cell killing was through both apoptotic and autophagic pathways. Induction of autophagy by curcumin, an autophagic stimulator, inhibited the expression of PD-L1 and enhanced cytolytic activity of NCL-specific T cells. These findings provide the potential clinical approaches targeting NCLHigh/PD-L1High TNBC cells with NCL-specific T cells in combination with a PD-L1 inhibitor or autophagic stimulator.
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Antígeno B7-H1 , Neoplasias de Mama Triplo Negativas , Transferência Adotiva , Anticorpos/farmacologia , Humanos , Fosfoproteínas , Proteínas de Ligação a RNA , Linfócitos T , Neoplasias de Mama Triplo Negativas/patologia , NucleolinaRESUMO
Cancer-associated fibroblasts (CAFs) are the dominant component of the tumor microenvironment (TME) that can be beneficial to the generation and progression of cancer cells leading to chemotherapeutic failure via several mechanisms. Nevertheless, the roles of CAFs on anti-cancer drug response need more empirical evidence in cholangiocarcinoma (CCA). Herein, we examined the oncogenic roles of CAFs on gemcitabine resistance in CCA cells mediated via IL-6/STAT3 activation. Our findings showed that CCA-derived CAFs promote cell viability and enhance gemcitabine resistance in CCA cells through the activation of IL-6/STAT3 signaling. High expression of IL-6R was correlated with a poor overall survival rate and gemcitabine resistance in CCA, indicating that IL-6R can be a prognostic or predictive biomarker for the chemotherapeutic response of CCA patients. Blockade of IL-6R on CCA cells by tocilizumab, an IL-6R humanized antihuman monoclonal antibody, contributed to inhibition of the CAF-CCA interaction leading to enhancement of gemcitabine sensitivity in CCA cells. The results of this study should be helpful for modifying therapeutic regimens aimed at targeting CAF interacting with cancer cells resulting in the suppression of the tumor progression but enhancement of drug sensitivity.
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BACKGROUND/AIM: The functions of interleukin 33 (IL-33) in cholangiocarcinoma (CCA) are unclear. This study aimed to evaluate the roles of IL-33 in CCA progression. MATERIALS AND METHODS: The effect of intracellular IL-33 using shIL-33 knocked down KKU-055 (IL-33KD-KKU-055) compared to parental (Pa) KKU-055 and extracellular IL-33 using recombinant human IL-33 (rhIL-33) treatment on the proliferation and invasion of CCA cells grown in 3D cultures was studied. Relevant markers were determined by western blot or ELISA. RESULTS: IL-33KD-KKU-055 cells showed increased proliferation and invasion in 3D cultures compared to Pa-KKU-055 cells, with NF-κB and IL-6 up-regulation. Treatment with 2 ng/ml rhIL-33 promoted Pa-KKU-055 cell proliferation by inducing NF-κB and IL-6 expressions. Upon GSK-3ß inactivation and increased nuclear full-length IL-33 (flIL-33), 20 ng/ml rhIL-33 had no effect on proliferation. Both 2 and 20 ng/ml rhIL-33 induced proliferation and invasion of IL-33-negative KKU-213 cells in 3D cultures, as well as NF-κB and IL-6 up-regulation. CONCLUSION: Intracellular and extracellular IL-33 have distinct roles in the mechanisms of CCA progression.
Assuntos
Neoplasias dos Ductos Biliares/prevenção & controle , Biomarcadores Tumorais/metabolismo , Colangiocarcinoma/prevenção & controle , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/metabolismo , Interleucina-33/farmacologia , NF-kappa B/metabolismo , Apoptose , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/patologia , Biomarcadores Tumorais/genética , Proliferação de Células , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patologia , Glicogênio Sintase Quinase 3 beta/genética , Humanos , NF-kappa B/genética , Invasividade Neoplásica , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Interleukin-6 (IL-6) released by cancer-associated fibroblasts (CAFs) has been shown to associate with the malignant behavior of cholangiocarcinoma (CCA). Here, we aimed to validate with clinical and molecular data the hypothesis that CAF infiltration and release of IL-6 predict poor prognosis in CCA patients following dysregulation of autophagy in cancer cells. METHODS: Stromal IL-6 and cancer-cell-associated autophagy proteins LC3 and p62 were assayed by Tissue MicroArray immunohistochemistry and their expression correlated with overall survival (OS) in a cohort of 70 CCA patients. The 5-FU cytotoxicity and autophagy were determined in CCA cells cultured with CAF-conditioned medium. RESULTS: We show that patients bearing a CCA with low production of stromal IL-6 and active autophagy flux in the cancer cells have the best prognosis and this correlates with a more effective response to post-operative chemotherapy. A similar trend was observed in CCA patients from the TCGA database. In vitro genetic manipulation of IL-6 production by primary CAFs isolated from human CCA showed that IL-6 impairs the autophagy-associated apoptotic response to 5-FU in human CCA cells. Stromal IL-6 inhibition of autophagy in cancer cells was confirmed in an animal model of CCA. CONCLUSION: Our data support a therapeutic strategy that includes autophagy-enhancing drugs along with adjuvants limiting the stromal inflammation (i.e., the secretion of IL-6) to improve the survival of CCA patients.
RESUMO
Background: Ovarian cancer progression and invasiveness are promoted by a range of soluble factors released by cancer cells and stromal cells within the tumor microenvironment. Our previous studies demonstrated that resveratrol (RV), a nutraceutical and caloric restriction mimetic with tumor-suppressive properties, counteracts cancer cell motility induced by stromal IL-6 by upregulating autophagy. Lysophosphatidic acid (LPA), a bioactive phospholipid that shows elevated levels in the tumor microenvironment and the ascites of ovarian cancers, stimulates the growth and tissue invasion of cancer cells. Whether LPA elicits these effects by inhibiting autophagy and through which pathway and whether RV can counteract the same remain obscure. Aims: To investigate the molecular pathways involved in LPA-induced ovarian cancer malignancy, particularly focusing on the role of autophagy, and the ability of RV to counteract LPA activity. Results: LPA stimulated while RV inhibited ovarian cancer cell migration. Transcriptomic and bioinformatic analyses showed an opposite regulation by LPA and RV of genes linked to epithelial-to-mesenchymal transition (EMT) and autophagy with involvement of the PI3K-AKT, JAK-STAT and Hedgehog (Hh) pathways. LPA upregulated the Hh and EMT members GLI1, BMI-1, SNAIL-1 and TWIST1 and inhibited autophagy, while RV did the opposite. Similar to the inhibitors of the Hh pathway, RV inhibited LPA-induced cancer cell migration and 3D growth of ovarian cancer cells. BMI-1 silencing prevented LPA-induced EMT, restored autophagy and hampered cell migration, resembling the effects of RV. TCGA data analyses indicated that patients with low expression of Hh/EMT-related genes together with active autophagy flux tended to have a better prognosis and this correlates with a more effective response to platinum therapy. In in vitro 3D spheroids, LPA upregulated BMI-1, downregulated autophagy and inhibited platinum toxicity while RV and Hh inhibitors restored autophagy and favored BAX-mediated cell death in response to platinum. Conclusions: By inhibiting the Hh pathway and restoration of autophagy, RV counteracts LPA-induced malignancy, supporting its inclusion in the therapy of ovarian cancer for limiting metastasis and chemoresistance.
Assuntos
Autofagia , Movimento Celular , Resistencia a Medicamentos Antineoplásicos , Proteínas Hedgehog/metabolismo , Neoplasias Ovarianas/patologia , Platina/farmacologia , Resveratrol/farmacologia , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Proteínas Hedgehog/genética , Humanos , Lisofosfolipídeos , Neoplasias Ovarianas/genética , Complexo Repressor Polycomb 1/metabolismo , Prognóstico , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transcriptoma/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
The tumor microenvironment composed of a mixture of stromal cells and their secretions has a marked impact on cancer progression. In particular, soluble factors and metabolites contribute to malignancy through the dysregulation of autophagy in cancer cells. The present study investigated the effects of ovarian cancerassociated fibroblasts (OVCAFs) with their secretory substances on the autophagy and migration of ovarian cancer cells. The conditionedmedium (CM) of OVCAFs isolated from fresh human ovarian cancer tissues was analyzed for the levels of 27 common cytokines/chemokines using a cytokine array. Autophagy in cancer cells was assessed by determining the expression of the vacuolar form of LC3 by western blot analysis and immunofluorescence. Cancer cell migration was assessed by Transwell migration assay. Interleukin (IL)8 was found to be the most highly upregulated cytokine among the cytokines/chemokines found in the OVCAFCM. The role of IL8 in ovarian cancer cell migration and its mechanistic link with autophagy was investigated. Recombinant human IL8 (rhIL8) stimulated the migration of SKOV3 and Kuramochi ovarian cancer cells, and concurrently downregulated basal autophagy, in concentrationdependent manner. Compared to the CM of control counterpart normal fibroblasts isolated from benign ovaries (OVNFCM), the CM from 3 OVCAF isolates (namely, OVCAF9, 20 and 43) exerted effects similar to rhIL8 on both cancer cell lines. The pharmacological induction of autophagy with rapamycin or metformin attenuated the promigratory effects of IL8. Neutralizing antiIL8 antibody counteracted the inhibitory effect of OVCAFCM on basal autophagy. On the whole, the present study highlights the involvement of IL8 released by CAFs in the ovarian tumor microenvironment in promoting cancer cell migration through the suppression of autophagy.
Assuntos
Fibroblastos Associados a Câncer/metabolismo , Interleucina-8/metabolismo , Neoplasias Ovarianas/metabolismo , Regulação para Cima , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Meios de Cultivo Condicionados/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-8/farmacologia , Metformina/farmacologia , Proteínas Associadas aos Microtúbulos/metabolismo , Sirolimo/farmacologia , Microambiente Tumoral , Regulação para Cima/efeitos dos fármacosRESUMO
Pyrvinium pamoate (PP), an FDA-approved anthelmintic drug, has been validated as a highly potent anti-cancer agent and patented recently as a potential chemotherapeutic drug for various cancers. The aims of this study were, therefore, to investigate the ability of PP in anti-proliferative activity and focused on the lipid profiles revealing the alteration of specific lipid species in the liver fluke Opisthorchis viverrini (Ov)-associated cholangiocarcinoma (CCA) cells. PP inhibited CCA cell viability through suppressing mitochondrial membrane potential (MMP) and ATP productions, leading to apoptotic cell death. Liquid chromatography-mass spectrometry combined with chemometrics was performed to investigate lipid alteration during PP-induced apoptosis. The lipidomic analyses showed the altered lipid signatures of CCA cell types including S-acetyldihydrolipoamide, methylselenopyruvate, and triglycerides that were increased in PP-treated CCA cells. In contrast, the levels of sphinganine and phosphatidylinositol were lower in the PP-treated group compared with its counterpart. The orthogonal partial-least squares regression analysis revealed that PP-induced MMP dysfunction, leading to remarkably reduced ATP level, was significantly associated with triglyceride (TG) accumulation observed in PP-treated CCA cells. Our findings indicate that PP could suppress the MMP function, which causes inhibition of CCA cell viability through lipid production, resulting in apoptotic induction in CCA cells. These findings provide an anti-cancer mechanism of PP under apoptotic induction ability that may serve as the alternative approach for CCA treatment.