RESUMO
BACKGROUND: Cancer-endothelial interplay is crucial for tumor behavior, yet the molecular mechanisms involved are largely unknown. Interleukin(IL)-30, which is expressed as a membrane-anchored cytokine by human prostate cancer (PC) cells, promotes PC vascularization and progression, but the underlying mechanisms have yet to be fully explored. METHODS: PC-endothelial cell (EC) interactions were investigated, after coculture, by flow cytometry, transcriptional profiling, western blot, and ELISA assays. Proteome profiler phospho-kinase array unveiled the molecular pathways involved. The role of tumor-derived IL30 on the endothelium's capacity to generate autocrine circuits and vascular budding was determined following IL30 overexpression, by gene transfection, or its deletion by CRISPR/Cas9 genome editing. Clinical value of the experimental findings was determined through immunopathological study of experimental and patient-derived PC samples, and bioinformatics of gene expression profiles from PC patients. RESULTS: Contact with PC cells favors EC proliferation and production of angiogenic and angiocrine factors, which are boosted by PC expression of IL30, that feeds autocrine loops, mediated by IGF1, EDN1, ANG and CXCL10, and promotes vascular budding and inflammation, via phosphorylation of multiple signaling proteins, such as Src, Yes, STAT3, STAT6, RSK1/2, c-Jun, AKT and, primarily CREB, GSK-3α/ß, HSP60 and p53. Deletion of the IL30 gene in PC cells inhibits endothelial expression of IGF1, EDN1, ANG and CXCL10 and substantially impairs tumor angiogenesis. In its interaction with IL30-overexpressing PC cells the endothelium boosts their expression of a wide range of immunity regulatory genes, including CCL28, CCL4, CCL5, CCR2, CCR7, CXCR4, IL10, IL13, IL17A, FASLG, IDO1, KITLG, TNFA, TNFSF10 and PDCD1, and cancer driver genes, including BCL2, CCND2, EGR3, IL6, VEGFA, KLK3, PTGS1, LGALS4, GNRH1 and SHBG. Immunopathological analyses of PC xenografts and in silico investigation of 1116 PC cases, from the Prostate Cancer Transcriptome Atlas, confirmed the correlation between the expression of IL30 and that of both pro-inflammatory genes, NOS2, TNFA, CXCR5 and IL12B, and cancer driver genes, LGALS4, GNRH1 and SHBG, which was validated in a cohort of 80 PC patients. CONCLUSIONS: IL30 regulates the crosstalk between PC and EC and reshapes their transcriptional profiles, triggering angiogenic, immunoregulatory and oncogenic gene expression programs. These findings highlight the angiostatic and oncostatic efficacy of targeting IL30 to fight PC.
Assuntos
Angiogênese , Neoplasias da Próstata , Humanos , Masculino , Linhagem Celular Tumoral , Endotélio/metabolismo , Endotélio/patologia , Galectina 4/metabolismo , Interleucinas , Neoplasias da Próstata/patologia , Transdução de SinaisRESUMO
BACKGROUND: Progression of colorectal cancer (CRC), a leading cause of cancer-related death worldwide, is driven by colorectal cancer stem cells (CR-CSCs), which are regulated by endogenous and microenvironmental signals. Interleukin (IL)-30 has proven to be crucial for CSC viability and tumor progression. Whether it is involved in CRC tumorigenesis and impacts clinical behavior is unknown. METHODS: IL30 production and functions, in stem and non-stem CRC cells, were determined by western blot, immunoelectron microscopy, flow cytometry, cell viability and sphere formation assays. CRISPR/Cas9-mediated deletion of the IL30 gene, RNA-Seq and implantation of IL30 gene transfected or deleted CR-CSCs in NSG mice allowed to investigate IL30's role in CRC oncogenesis. Bioinformatics and immunopathology of CRC samples highlighted the clinical implications. RESULTS: We demonstrated that both CR-CSCs and CRC cells express membrane-anchored IL30 that regulates their self-renewal, via WNT5A and RAB33A, and/or proliferation and migration, primarily by upregulating CXCR4 via STAT3, which are suppressed by IL30 gene deletion, along with WNT and RAS pathways. Deletion of IL30 gene downregulates the expression of proteases, such as MMP2 and MMP13, chemokine receptors, mostly CCR7, CCR3 and CXCR4, and growth and inflammatory mediators, including ANGPT2, CXCL10, EPO, IGF1 and EGF. These factors contribute to IL30-driven CR-CSC and CRC cell expansion, which is abrogated by their selective blockade. IL30 gene deleted CR-CSCs displayed reduced tumorigenicity and gave rise to slow-growing and low metastatic tumors in 80% of mice, which survived much longer than controls. Bioinformatics and CIBERSORTx of the 'Colorectal Adenocarcinoma TCGA Nature 2012' collection, and morphometric assessment of IL30 expression in clinical CRC samples revealed that the lack of IL30 in CRC and infiltrating leucocytes correlates with prolonged overall survival. CONCLUSIONS: IL30 is a new CRC driver, since its inactivation, which disables oncogenic pathways and multiple autocrine loops, inhibits CR-CSC tumorigenicity and metastatic ability. The development of CRISPR/Cas9-mediated targeting of IL30 could improve the current therapeutic landscape of CRC.
Assuntos
Neoplasias do Colo , Neoplasias Colorretais , Camundongos , Animais , Neoplasias do Colo/patologia , Linhagem Celular Tumoral , Edição de Genes , Sistemas CRISPR-Cas/genética , Proliferação de Células , Células-Tronco Neoplásicas/patologia , Neoplasias Colorretais/patologia , Transformação Celular Neoplásica/patologia , Carcinogênese/genética , Interleucinas/genéticaRESUMO
BACKGROUND: Metastatic prostate cancer (PC) is a leading cause of cancer death in men worldwide. Targeting of the culprits of disease progression is an unmet need. Interleukin (IL)-30 promotes PC onset and development, but whether it can be a suitable therapeutic target remains to be investigated. Here, we shed light on the relationship between IL30 and canonical PC driver genes and explored the anti-tumor potential of CRISPR/Cas9-mediated deletion of IL30. METHODS: PC cell production of, and response to, IL30 was tested by flow cytometry, immunoelectron microscopy, invasion and migration assays and PCR arrays. Syngeneic and xenograft models were used to investigate the effects of IL30, and its deletion by CRISPR/Cas9 genome editing, on tumor growth. Bioinformatics of transcriptional data and immunopathology of PC samples were used to assess the translational value of the experimental findings. RESULTS: Human membrane-bound IL30 promoted PC cell proliferation, invasion and migration in association with STAT1/STAT3 phosphorylation, similarly to its murine, but secreted, counterpart. Both human and murine IL30 regulated PC driver and immunity genes and shared the upregulation of oncogenes, BCL2 and NFKB1, immunoregulatory mediators, IL1A, TNF, TLR4, PTGS2, PD-L1, STAT3, and chemokine receptors, CCR2, CCR4, CXCR5. In human PC cells, IL30 improved the release of IGF1 and CXCL5, which mediated, via autocrine loops, its potent proliferative effect. Deletion of IL30 dramatically downregulated BCL2, NFKB1, STAT3, IGF1 and CXCL5, whereas tumor suppressors, primarily SOCS3, were upregulated. Syngeneic and xenograft PC models demonstrated IL30's ability to boost cancer proliferation, vascularization and myeloid-derived cell infiltration, which were hindered, along with tumor growth and metastasis, by IL30 deletion, with improved host survival. RNA-Seq data from the PanCancer collection and immunohistochemistry of high-grade locally advanced PCs demonstrated an inverse association (chi-squared test, p = 0.0242) between IL30 and SOCS3 expression and a longer progression-free survival of patients with IL30NegSOCS3PosPC, when compared to patients with IL30PosSOCS3NegPC. CONCLUSIONS: Membrane-anchored IL30 expressed by human PC cells shares a tumor progression programs with its murine homolog and, via juxtacrine signals, steers a complex network of PC driver and immunity genes promoting prostate oncogenesis. The efficacy of CRISPR/Cas9-mediated targeting of IL30 in curbing PC progression paves the way for its clinical use.
Assuntos
Antígeno B7-H1 , Neoplasias da Próstata , Animais , Antígeno B7-H1/genética , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proliferação de Células , Quimiocina CXCL5/genética , Quimiocina CXCL5/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Humanos , Fator de Crescimento Insulin-Like I , Interleucinas/metabolismo , Masculino , Camundongos , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Receptores de Quimiocinas , Proteína 3 Supressora da Sinalização de Citocinas/genética , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismoRESUMO
Colorectal cancer (CRC) is one of the most common cancer worldwide, with a growing impact on public health and clinical management. Immunotherapy has shown promise in the treatment of advanced cancers, but needs to be improved for CRC, since only a limited fraction of patients is eligible for treatment, and most of them develop resistance due to progressive immune exhaustion. Here, we identify the transcriptional, molecular, and cellular traits of the immune exhaustion associated with CRC and determine their relationships with the patient's clinic-pathological profile. Bioinformatic analyses of RNA-sequencing data of 594 CRCs from TCGA PanCancer collection, revealed that, in the wide range of immune exhaustion genes, those coding for PD-L1, LAG3 and T-bet were associated (Cramér's V=0.3) with MSI/dMMR tumors and with a shorter overall survival (log-rank test: p=0.0004, p=0.0014 and p=0.0043, respectively), whereas high levels of expression of EOMES, TRAF1, PD-L1, FCRL4, BTLA and SIGLEC6 were associated with a shorter overall survival (log-rank test: p=0.0003, p=0.0188, p=0.0004, p=0.0303, p=0.0052 and p=0.0033, respectively), independently from the molecular subtype of CRC. Expression levels of PD-L1, PD-1, LAG3, EOMES, T-bet, and TIGIT were significantly correlated with each other and associated with genes coding for CD4+ and CD8+CD3+ T cell markers and NKp46+CD94+EOMES+T-bet+ cell markers, (OR >1.5, p<0.05), which identify a subset of group 1 innate lymphoid cells, namely conventional (c)NK cells. Expression of TRAF1 and BTLA co-occurred with both T cell markers, CD3γ, CD3δ, CD3ε, CD4, and B cell markers, CD19, CD20 and CD79a (OR >2, p<0.05). Expression of TGFß1 was associated only with CD4+ and CD8+CD3ε+ T cell markers (odds ratio >2, p<0.05). Expression of PD-L2 and IDO1 was associated (OR >1.5, p<0.05) only with cNK cell markers, whereas expression of FCRL4, SIGLEC2 and SIGLEC6 was associated (OR >2.5; p<0.05) with CD19+CD20+CD79a+ B cell markers. Morphometric examination of immunostained CRC tissue sections, obtained from a validation cohort of 53 CRC patients, substantiated the biostatistical findings, showing that the highest percentage of immune exhaustion gene expressing cells were found in tumors from short-term survivors and that functional exhaustion is not confined to T lymphocytes, but also involves B cells, and cNK cells. This concept was strengthened by CYBERSORTx analysis, which revealed the expression of additional immune exhaustion genes, in particular FOXP1, SIRT1, BATF, NR4A1 and TOX, by subpopulations of T, B and NK cells. This study provides novel insight into the immune exhaustion landscape of CRC and emphasizes the need for a customized multi-targeted therapeutic approach to overcome resistance to current immunotherapy.
Assuntos
Linfócitos B/imunologia , Neoplasias Colorretais/imunologia , Células Matadoras Naturais/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos T/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos B/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Colo/imunologia , Colo/patologia , Colo/cirurgia , Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/terapia , Biologia Computacional , Conjuntos de Dados como Assunto , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Imunidade Inata/genética , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Mucosa Intestinal/cirurgia , Estimativa de Kaplan-Meier , Células Matadoras Naturais/metabolismo , Contagem de Linfócitos , Linfócitos do Interstício Tumoral/metabolismo , Masculino , Pessoa de Meia-Idade , RNA-Seq , Reto/imunologia , Reto/patologia , Reto/cirurgia , Linfócitos T/metabolismo , Fatores de TempoRESUMO
Breast cancer (BC) mortality is mainly due to metastatic disease, which is primarily driven by cancer stem cells (CSC). The chemokine C-X-C motif ligand-1 (CXCL1) is involved in BC metastasis, but the question of whether it regulates breast cancer stem cell (BCSC) behavior is yet to be explored. Here, we demonstrate that BCSCs express CXCR2 and produce CXCL1, which stimulates their proliferation and self-renewal, and that CXCL1 blockade inhibits both BCSC proliferation and mammosphere formation efficiency. CXCL1 amplifies its own production and remarkably induces both tumor-promoting and immunosuppressive factors, including SPP1/OPN, ACKR3/CXCR7, TLR4, TNFSF10/TRAIL and CCL18 and, to a lesser extent, immunostimulatory cytokines, including IL15, while it downregulates CCL2, CCL28, and CXCR4. CXCL1 downregulates TWIST2 and SNAI2, while it boosts TWIST1 expression in association with the loss of E-Cadherin, ultimately promoting BCSC epithelial-mesenchymal transition. Bioinformatic analyses of transcriptional data obtained from BC samples of 1,084 patients, reveals that CXCL1 expressing BCs mostly belong to the Triple-Negative (TN) subtype, and that BC expression of CXCL1 strongly correlates with that of pro-angiogenic and cancer promoting genes, such as CXCL2-3-5-6, FGFBP1, BCL11A, PI3, B3GNT5, BBOX1, and PTX3, suggesting that the CXCL1 signaling cascade is part of a broader tumor-promoting signaling network. Our findings reveal that CXCL1 functions as an autocrine growth factor for BCSCs and elicits primarily tumor progression and immune escape programs. Targeting the CXCL1/CXCR2 axis could restrain the BCSC compartment and improve the treatment of aggressive BC.
RESUMO
BACKGROUND: Breast cancer (BC) progression to metastatic disease is the leading cause of death in women worldwide. Metastasis is driven by cancer stem cells (CSCs) and signals from their microenvironment. Interleukin (IL) 30 promotes BC progression, and its expression correlates with disease recurrence and mortality. Whether it acts by regulating BCSCs is unknown and could have significant therapeutic implications. METHODS: Human (h) and murine (m) BCSCs were tested for their production of and response to IL30 by using flow cytometry, confocal microscopy, proliferation and sphere-formation assays, and PCR array. Immunocompetent mice were used to investigate the role of BCSC-derived IL30 on tumor development and host outcome. TCGA PanCancer and Oncomine databases provided gene expression data from 1084 and 75 hBC samples, respectively, and immunostaining unveiled the BCSC microenvironment. RESULTS: hBCSCs constitutively expressed IL30 as a membrane-anchored glycoprotein. Blocking IL30 hindered their proliferation and self-renewal efficiency, which were boosted by IL30 overexpression. IL30 regulation of immunity gene expression in human and murine BCSCs shared a significant induction of IL23 and CXCL10. Both immunoregulatory mediators stimulated BCSC proliferation and self-renewal, while their selective blockade dramatically hindered IL30-dependent BCSC proliferation and mammosphere formation. Orthotopic implantation of IL30-overexpressing mBCSCs, in syngeneic mice, gave rise to poorly differentiated and highly proliferating MYC+KLF4+LAG3+ tumors, which expressed CXCL10 and IL23, and were infiltrated by myeloid-derived cells, Foxp3+ T regulatory cells and NKp46+RORγt+ type 3 innate lymphoid cells, resulting in increased metastasis and reduced survival. In tumor tissues from patients with BC, expression of IL30 overlapped with that of CXCL10 and IL23, and ranked beyond the 95th percentile in a Triple-Negative enriched BC collection from the Oncomine Platform. CIBERSORTx highlighted a defective dendritic cell, CD4+ T and γδ T lymphocyte content and a prominent LAG3 expression in IL30highversus IL30low human BC samples from the TCGA PanCancer collection. CONCLUSIONS: Constitutive expression of membrane-bound IL30 regulates BCSC viability by juxtacrine signals and via second-level mediators, mainly CXCL10 and IL23. Their autocrine loops mediate much of the CSC growth factor activity of IL30, while their paracrine effect contributes to IL30 shaping of immune contexture. IL30-related immune subversion, which also emerged from computational analyses, strongly suggests that targeting IL30 can restrain the BCSC compartment and counteract BC progression.