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

Abstract

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.

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

“…Primarily found in high-grade and stage of PC, IL30 expression is associated with a thriving vasculature, immune suppression, and tumor progression [ 14 – 16 ]. To assess its potential impact on the PC-EC crosstalk, we used two human cell lines derived from the metastases of high-grade PCs, and that express membrane-anchored IL30 [ 15 ], namely DU145 cells [ 19 ], endowed with a CK8/14 + AR + PSA + phenotype, and castration resistant PC3 cells [ 20 ], endowed with a CD44 + AR − PSA − CgA + NSE + neuroendocrine phenotype. Both cell lines were engineered to overexpress IL30, henceforth referred to as IL30-DU145 and IL30-PC3 cells or were knocked out (KO) for the IL30 gene by CRISPR/Cas9 genome editing, hereinafter referred to as IL30KO-DU145 and IL30KO-PC3 cells [ 15 ].…”

“…To assess its potential impact on the PC-EC crosstalk, we used two human cell lines derived from the metastases of high-grade PCs, and that express membrane-anchored IL30 [ 15 ], namely DU145 cells [ 19 ], endowed with a CK8/14 + AR + PSA + phenotype, and castration resistant PC3 cells [ 20 ], endowed with a CD44 + AR − PSA − CgA + NSE + neuroendocrine phenotype. Both cell lines were engineered to overexpress IL30, henceforth referred to as IL30-DU145 and IL30-PC3 cells or were knocked out (KO) for the IL30 gene by CRISPR/Cas9 genome editing, hereinafter referred to as IL30KO-DU145 and IL30KO-PC3 cells [ 15 ]. Since the tumor vascular bed stems from the activation and proliferation of normal pre-existing vessels, for the PC-EC coculture experiments, we used primary ECs derived from human umbilical vein, HUVEC, and immortalized ECs isolated from human aortic endothelia, TeloHAEC, hereinafter referred to as HAEC.…”

“…Primary human umbilical vein endothelial cells (HUVEC; #PCS-100–010) and immortalized human aortic endothelial cells (TeloHAEC; RRID:CVCL_Z065) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were cultivated in Vascular Cell Basal Medium (#PCS-100–030; ATCC) plus Endothelial Cell Growth Kit-VEGF (#PCS-100–041; ATCC). Human PC cell lines, AR − PC3 and AR + DU145 [ 15 , 19 , 20 ], were purchased from the ATCC, which authenticated them by short tandem repeat profile analysis. PC cells were cultured in RPMI-1640 (#R8758; Merck, Darmstadt, Germany) with 10% fetal calf serum (#F1283; Merck).…”

“…The CRISPR/Cas9 technology was used to generate IL30 gene knockout (IL30KO) DU145 and PC3 cells, as we described in ref.15. IL30 gene knockout was validated by WB [ 15 ].…”

“…We recently demonstrated that the immunoregulatory molecule, interleukin(IL)-30 (IL27/p28) [ 13 ], which is expressed as a membrane-anchored cytokine, by human PC cells, and in the microenvironment, by tumor-infiltrating immune cells, mainly macrophages and myeloid-derived suppressor cells (MDSCs) [ 14 , 15 ], plays a critical role in PC onset and progression, by triggering a cascade of proinflammatory and oncogenic events, in association with the development of a robust vascular network [ 16 , 17 ]. A rich vascular supply has been described in IL30-overexpressing prostatic and mammary tumors, in both human and murine models of cancer, by contrast, a poor vascularization characterized the slow growing IL30-deficient tumors [ 15 – 18 ].…”