LATS1 Knockdown with SiRNA Induces Pyroptosis via the Activation of TNF-α/NF-κB Signaling in Bladder Cancer Cells

Abstract

Background: Traditionally, it is believed that large tumor suppressor 1 (LATS1) is a negative regulator of oncogene. But the latest research showed that LATS1 has an opposite effect in some tumors. We found that LATS1 has a cancer-promoting effect in BLCA, but the specific mechanism is unknown. Methods: RNAi method was used for the related genetic functional analysis. CCK-8 method and colony formation assay were used to explore the cellular viabilities and proliferation of BLCA cells transfected with LATS1 siRNAs (si-LATS1), in vitro. Flow Cytometry (FCM) was used to analyze the cell cycle and cellular apoptosis of the BLCA cells or the expression of CD68, CD86, CD11b, and CD163 in PMA-treated THP-1 macrophages incubated with conditioned medium (CM) from the si-LATS1 cells or in the THP-1 macrophages cultured directly with BLCA cells. RT-qPCR method was used to detect the mRNA levels of IL-1β, IL-2/4/6/10/18, TNF-α, and IFN-γ in the BLCA cells. Western Blot was performed to detect the expressions of LATS1, Yap1, Bcl-2, Bax, caspase-1/3, GSDMD, TNF-α IL-1β, IL-18, NF-κB, AIM2, NLRC4 and NLRP3 in the BLCA cell lines. For in vivo experiments, a xenograft model was used to investigate the inhibitory effects of LATS1 knockdown on BLCA cells in nude mice.Results: LATS1 knockdown via siRNA inhibited the proliferation of the BLCA cells, neither changing cell cycle distribution nor inducing apoptosis. Via further analysis, we found that the expressions of TNF-α, p-NF-κB/RelA, NLRP3, NLRC4, and AIM2 in si-LATS1 BLCA cells were significantly increased. The activity of caspase-1 and the expressions of IL-1β, IL-18 and GSDMD were obviously increased in the si-LATS1 BLCA cells, which was restored by the addition of NF-κB inhibitor PS341. In the LATS1 over-expression cells (OE-LATS1), the expressions of TNF-α, p-NF-κB/p65, NLRP3, NLRC4 and AIM2 were notably inhibited, and the expressions of IL-1β, IL-18 and GSDMD were significantly decreased. THP-1 macrophages exhibited an M1 phenotype polarization in the presence of si-LATS1 BUC-87 cell supernatant or when cocultured with the BLCA cells transfected with LATS1 siRNA, represented by an increase in the surface expression of CD86. Furthermore, we observed that LATS1 knockdown inhibited the cell proliferation in xenograft model. Conclusion: Our findings showed that LATS1 knockdown via siRNA induces BLCA cell pyrolysis due to the enhanced formation of inflammasomes by activation of TNF-α/NF-κB pathway; and inflammatory factors released by the pyrolytic cells promote M1 polarization of THP-1-derived macrophages in vitro, providing a therapeutic target for BLCA and a brand-new idea for the development of BLCA immunotherapy drugs.

Non-apoptotic cell death-based cancer therapy: Molecular mechanism, pharmacological modulators, and nanomedicine

“…In recent years, many drugs such as simvastatin and chalcone analogues have been found to mediate pyroptosis of cancer cells through the caspase1/GSDMD pathway or the caspase3/GSDME pathway 139 , 140 . For example, dipeptidyl peptidase DPP8 and DPP9 (DPP8/9) inhibitors can activate Nlrp1b in the inflammatory body sensor NLR family, and induce pyroptosis of acute myeloid leukemia (AML) cell line through caspase-1 pathway 141 . Simvastatin activates caspase-1-dependent GSDMD cleavage by activating NLRP3 to induce pyroptosis of H1299 and A549 cells.…”