p62 mRNA suppresses NLRP1 expression in cutaneous SCC cells through miR-34a-5p

Abstract

The inflammasome sensor NLRP1 is mainly expressed by epithelial cells including keratinocytes of human skin. Germline gain-of-function mutations in NLRP1 cause inflammatory skin syndromes and predispose patients to the development of cutaneous squamous cell carcinomas (cSCCs), a major type of skin cancer originating from keratinocytes. However, expression of NLRP1 is strongly reduced in cSCCs suggesting a complex role of the NLRP1 inflammasome in the development of this type of skin cancer. Suppression of NLRP1 expression in SCC cells is partially caused by an increase in p62 (SQSTM1), a cargo receptor for autophagy-dependent protein degradation. p62 is upregulated in numerous types of cancer and plays key roles in tumor development by activating different pathways. Here, we characterized the molecular mechanisms underlying suppression of NLRP1 expression by p62 in cSCCs. In SCC cells, NLRP1 activation is rescued by a knockdown or knockout of p62 mRNA and, consequently, protein expression, rather than by a knockout of p62 protein expression only. As these experiments suggest a regulation of NLRP1 by the p62 mRNA, we characterized p62 mRNA-regulated gene expression in SCC cells through RNA sequencing. In addition to mRNAs, we identified several differentially regulated microRNAs (miRs), including miR-34a-5p. These short non-coding RNAs regulate the stability or translation of mRNAs in a dynamic manner and a single miR can target multiple mRNAs. miR-34a-5p is an established tumor suppressor in different types of cancer and its expression is also downregulated in cSCCs. Although miR-34a-5p seems to bind neither p62 nor NLRP1 mRNA directly, it increases NLRP1 expression, most likely through an indirect and complex mechanism, which occurs at the RNA level. In summary, our findings revealed a novel pathway regulating suppression of the inflammasome sensor NLRP1 in SCC cells by p62, which occurs at the mRNA level and is mediated by miRs, including the tumor suppressive miR-34a-5p. Therefore, a pharmacological increase in miR-34a expression represents a treatment option for cSCC patients that allows not only to target know proteins regulated by miR-34a but also a reconstitution of NLRP1 expression.

Fig. 1. p62 mRNA, rather than protein, regulates inflammasome activation and expression of NLRP1.

A–D Expression of p62 was suppressed by different techniques to assess its impact on NLRP1 inflammasome activation. p62 protein expression was determined by western blot and IL-1β secretion, reflecting inflammasome activation, by ELISA 6 h after UVB irradiation (HPKs and SCCs) or nigericin treatment (THP-1 cells). A CRISPR/Cas9 knockout (p62.1 - protein only) of p62, (B) siRNA-mediated knockdown (mRNA and protein), or (C) CRISPR/dCas9-KRAB knockout (p62.2 K - mRNA and protein) in SCC12 cells, HPKs and THP-1 cells. D mRNA levels of NLRP1 and SQSTM1 (p62) upon protein (p62.1) or mRNA/protein (p62.2 K) knockout in SCC12 cells determined by qPCR in mock-treated cells. Data are represented as mean ± SD of three experiments using two-tailed unpaired t-test (N = 3) (A–C) or one-way analysis of variance with Dunnett’s multiple comparison test (N = 10) (D). (****P < 0.001, ***P ≤ 0.001, **P ≤ 0.01, and *P ≤ 0.05, ns = not significant). SCC squamous cell carcinoma, HPK human primary keratinocyte, THP-1 monocyte cell line.

Fig. 2. p62 mRNA, but not protein, regulates expression of other mRNAs and miRNAs.

A–D RNA sequencing data showing influence of p62 mRNA knockout on gene (A, C) and miRNA (B, D) expression. Differential expression analysis of RNA-seq data from SCC12 cells with a protein (p62.1) or mRNA (p62.2 K) knockout represented by Volcano plot (each dot represents one gene) and Heatmap showing differentially expressed genes or mature miRNAs. E Principal component analysis (PCA) and (F) non-metric multidimensional scaling (NMDS) (blue for RNA knockout, red and green for control and protein only knockout, respectively), highlighting distinct gene expression patterns. DOWN (downregulated genes/ miRNAs), NOT DE (mRNA/miRNA that were not significantly regulated in the screen), UP (upregulated genes/ miRNAs); N = 4.

Fig. 3. Validation of p62 mRNA-regulated genes (A) and miRNAs (B) using qPCR.

A, B SCC12 cells were transduced with a control (ctr.) gRNA or two different sgRNAs targeting p62 protein (p62.1), or mRNA (p62.2 K). Levels of mRNA (A) of the indicated genes and miRNAs (B) in mock-treated cells were determined by qPCR. Data are represented as mean ± SD of N = 10 (A) and N = 5 (B), p values are visualized using one-way analysis of variance with Dunnett’s multiple comparison test. (****P < 0.001, ***P ≤ 0.001, **P ≤ 0.01, and *P ≤ 0.05, ns = not significant).

Fig. 4. miR-34a-5p regulates NLRP1 expression.

A, B HPKs from two different donors were compared with SCC12 and SCC13 cells. A mRNA levels of SQSTM1 (p62), NLRP1, or (B) miRNA34a-5p were determined via qPCR. (C, D) HPKs (N = 2) and SCC12 cells (N = 3) were transfected with control (ctr.) antagomir or miR-34a-5p antagomir or treated with lipofectamine only, and analyzed using qPCR. A–D Data are represented as mean ± SD of three experiments (A, B) where two donors in the group of HPKs (N = 6) and two SCC cell lines (SCC12, SCC13) in the group of SCCs (N = 6) were subjected to two-tailed unpaired t-test with Welch’s correction or (C, D) one-way analysis of variance with Dunnett’s multiple comparison test. (****P < 0.001, ***P ≤ 0.001, **P ≤ 0.01, and *P ≤ 0.05, ns = not significant). SCC squamous cell carcinoma, HPK human primary keratinocyte, lipo lipofectamine.

Fig. 5. Graphical Summary.

In healthy skin, keratinocytes express high levels of NLRP1 and of the tumor suppressor miR-34a, while p62 expression remains relatively low. In cutaneous squamous cell carcinoma (cSCC) tumor cells, the expression of NLRP1 and miR-34a is decreased, while p62 expression is elevated. p62 mRNA negatively regulates miR-34a, while miR-34a positively regulates NLRP1 mRNA expression. Consequently, the increased expression of p62 in SCC cells leads to the suppression of NLRP1 expression, mediated by a decrease in miR-34a. Most likely, miR-34a does directly interact neither with p62 mRNA nor with NLRP1 mRNA.