The C-X-C Motif Chemokine Ligand 1 Sustains Breast Cancer Stem Cell Self-Renewal and Promotes Tumor Progression and Immune Escape Programs

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

CRISPR/Cas9-mediated deletion of Interleukin-30 suppresses IGF1 and CXCL5 and boosts SOCS3 reducing prostate cancer growth and mortality

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 IL30(Neg)SOCS3(Pos)PC, when compared to patients with IL30(Pos)SOCS3(Neg)PC. 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

Additional file 1 of Interleukin-30 subverts prostate cancer-endothelium crosstalk by fostering angiogenesis and activating immunoregulatory and oncogenic signaling pathways

Additional file 1: Supplementary Fig. S1. Cytofluorimetric analyses of endothelial cell marker expression in HUVEC (top of the panel), and HAEC (bottom of the panel). Both endothelial cell types expressed CD31/PECAM-1, CD34, CD54, CD309/VEGFR2 and vWF, but did not express CD45. Red lines: isotype control. Experiments were performed in triplicate. Supplementary Fig. S2. Western blot analysis showing that both HUVEC and HAEC express EBI3, but not the p28 (IL30) subunit of the heterodimeric (p28/EBI3) cytokine IL27. A representative image of triplicate experiments is shown. Supplementary Fig. S3. Cytofluorimetric analysis of cell apoptosis, by annexin V staining, in HUVEC (top of the panel) and HAEC (bottom of the panel) co-cultured or not with wild type DU145, IL30KO-DU145 or IL30-DU145. The negative annexin V staining indicates the absence of cells undergoing apoptosis in all conditions. Experiments were performed in triplicate. Supplementary Fig. S4. Western blot analysis of ANG, CXCL9, EDN1 and TGFB2 protein expression in HAEC co-cultured with DU145 (left side of the panel) or PC3 cells (right side of the panel). Representative images of three experiments. Supplementary Fig. S5. Western blot analysis of phosphorylated HSP60, p53, CREB and GSK3b proteins in ECs (HUVEC and HAEC) treated with rhIL30. Supplementary Fig. S6. Expression of CXCR3 isoforms in HAEC and HUVEC, as determined by RT-PCR. CXCR3A: 111 bp; CXCR3B: 79 bp; CXCR3-alt: 135 bp. Supplementary Fig. S7. Western blot analysis of CXCL6 and THBS2 protein expression in ECs (HAEC) co-cultured with IL30KO-DU145 (A) or IL30KO-PC3 (B) versus ECs co-cultured with control (NTgRNA-treated) or wild type (WT) DU145 or PC3 cells. Western blot analysis of IGF1 protein expression in ECs co-cultured with IL30 overexpressing or knockout DU145 or PC3 cells (C). Representative images of experiments in triplicate. Supplementary Fig. S8. Immunohistochemical analyses of PC3 (A) and DU145 (B) tumors show that expression of LGALS4 (a, b) and TNFa (c, d) is stronger in IL30 overexpressing tumors when compared to the respective wild type tumors (e, f). Results from EV-tumors were comparable to wild type tumors. Magnification: X400. Supplemental Fig. S9. Expression of CXCR5 in PC tissues obtained from patients bearing IL30Neg PC or IL30Pos PC. Inset shows a magnification of CXCR5 positive tumor cells. Representative images of the immunohistochemical study are shown. Magnification: X400. Supplementary Table S1. Antibodies used in flow cytometry. Supplementary Table S2. List of genes included in the apoptotic signaling pathway. Supplementary Table S3. Antibodies used in immunostaining. Supplementary Table S4. Gene list of the RT2 Profiler Human Angiogenesis PCR Array (#PAHS-024ZR). Supplementary Table S5. Immunohistochemical analysis of IL30 expression, microvessel density and proliferation index in wild type, IL30 gene transfected or deleted tumors and control EV-transfected or NTgRNA-treated tumors. Supplementary Table S6. Immunohistochemical analysis of inflammation and immunity genes in wild type and IL30 gene knockout tumors. Supplementary Table S7. Gene list of the RT2 Profiler Human Prostate Cancer PCR Array (#PAHS-135ZR). Supplementary Table S8. Gene list of the RT2 Profiler Human Cancer Inflammation & Immunity Crosstalk PCR Array (#PAHS-181Z). Supplementary Table S9. Immunohistochemical analysis of inflammation and immunity genes and prostate cancer driver genes in wild type and IL30 gene transfected tumors. Supplementary Table S10. Morphometric evaluation of IL30 and immunoregulatory and prostate cancer driver gene expression in prostate cancer samples from patients of the validation cohort*