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Cell adhesion tunes inflammatory TPL2 kinase signal transduction

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Abstract

Signaling through adhesion-related molecules is important for cancer growth and metastasis and cancer cells are resistant to anoikis, a form of cell death ensued by cell detachment from the extracellular matrix. Herein, we report that detached carcinoma cells and immortalized fibroblasts display defects in TNF and CD40 ligand (CD40L)-induced MEK-ERK signaling. Cell detachment results in reduced basal levels of the MEK kinase TPL2, compromises TPL2 activation and sensitizes carcinoma cells to death-inducing receptor ligands, mimicking the synthetic lethal interactions between TPL2 inactivation and TNF or CD40L stimulation. Focal Adhesion Kinase (FAK), which is activated in focal adhesions and mediates anchorage-dependent survival signaling, was found to sustain steady state TPL2 protein levels and to be required for TNF-induced TPL2 signal transduction. We show that when FAK levels are reduced, as seen in certain types of malignancy or malignant cell populations, the formation of cIAP2:RIPK1 complexes increases, leading to reduced TPL2 expression levels by a dual mechanism: first, by the reduction in the levels of NF-κΒ1 which is required for TPL2 stability; second, by the engagement of an RelA NF-κΒ pathway that elevates interleukin-6 production, leading to activation of STAT3 and its transcriptional target SKP2 which functions as a TPL2 E3 ubiquitin ligase. These data underscore a new mode of regulation of TNF family signal transduction on the TPL2-MEK-ERK branch by adhesion-related molecules that may have important ramifications for cancer therapy.

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Acknowledgements

We would like to thank Dr. George Gouridis (Foundation of Research & Technology Hellas, Crete) for critical reading of the manuscript, useful discussions, as well as or providing reagents, antibodies and expertise on protein biochemistry when needed. We would like to thank all current and previous members of Eliopoulos laboratory for sharing protocols, reagents, expertise and for useful discussions.

Funding

The project was realized using funds from European Commission (EC) Research Program INFLA-CARE (EC Contract 223151) granted to A.G.E.

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Authors and Affiliations

  1. Division of Basic Sciences, University of Crete Medical School, Heraklion, Greece

    Maria Vougioukalaki

  2. Institute for Molecular Biology and Biotechnology, Foundation of Research and Technology Hellas, Heraklion, Greece

    Maria Vougioukalaki & Aristides G. Eliopoulos

  3. Department of Biology, School of Medicine, National and Kapodistrian University of Athens, Athens, Greece

    Konstantina Georgila & Aristides G. Eliopoulos

  4. Center of Basic Research, Biomedical Research Foundation of the Academy of Athens, Athens, Greece

    Emmanouil I. Athanasiadis & Aristides G. Eliopoulos

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  1. Maria Vougioukalaki
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  2. Konstantina Georgila
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  3. Emmanouil I. Athanasiadis
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  4. Aristides G. Eliopoulos
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Contributions

MV: conceptualization, investigation, data collection and analysis, preparation of the original draft manuscript. K.G.: investigation, data collection and analysis; E.I.A: investigation, data collection and analysis; AGE: conceptualization, investigation and preparation of the final version of the paper. All authors critically reviewed and approved the final version.

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Correspondence to Aristides G. Eliopoulos.

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18_2022_4130_MOESM1_ESM.pdf

Legends for Supplementary Figures. Supporting Information, Fig. 1 Cell detachment does not affect TNF-induced JNK activation or EGF-stimulated ERK phosphorylation. (A) HeLa cervical carcinoma cells were detached from the culture plate and incubated in suspension for 3 hrs prior to either re-plating (adherent, ADH) or continuation of culture under detached (DET) conditions for another hour. Cells were then stimulated with 50 ng/ml TNF for various time intervals as indicated before lysis and assessment of JNK phosphorylation (p-JNK1/2) or β-Actin by immunoblot. (B) HeLa cells were cultured as above and were stimulated with 100 ng/mL EGF prior to lysis and analysis of p-ERK levels by immunoblot. Supporting Information, Fig. 2 The contribution of TNFR1 signaling components to TNF-mediated ERK activation. HeLa cells were transfected with siRNAs targeting TNFR1 signaling components RIPK1, cIAP1/2, CYLD, TPL2, PTK2, TRAF2 prior to stimulation with 50 ng/ml TNF for 10 min. ERK phosphorylation was assessed and compared to control siRNA-transfected cultures. Supporting Information, Fig. 3 Specificity of the effect of PTK2 knockdown on ERK phosphorylation. HeLa cells were transfected with human PTK2 siRNA and next day, MYC epitope tagged (MT) chicken FAK was co-transfected with HA-ERK1. Twenty four hours later cells were stimulated with 50ng/ml TNF or left untreated, lysates were immunoprecipitated with anti-HA and immunoblotted for p-ERK or ERK1. Supporting Information, Fig. 4 Relative increases in pSTAT3 levels in HeLa cells (CNT) cultured with supernatants (S/N) from siPTK2 versus control siRNA (siLuc)-transfected cells for 4 or 8 hrs prior, related to Fig. 6E. Two-way ANOVA was used to assess statistically significant differences (*p<0.05, **p<0.01, ***p<0.001). Supporting Information, Fig. 5 The knockdown of PTK2 in A549 lung adenocarcinoma cells increases the nuclear levels of RelA (A) and NF-κΒ transcriptional activity (B), assessed by luciferase reporter assays as described in the Materials and Methods. C; cytoplasmic, N; nuclear protein extracts. Supporting Information, Fig. 6 HeLa and A549 carcinoma cell bearing reduced levels of FAK are sensitive to SMAC mimetic (SM) LBW242-induced death. Cells were transfected with either siPTK2 (red lines) or control siRNA (black lines) and exposed to increasing doses of LBW242, as indicated. Viability was assessed 24 hours later by MTT assays. The data are representative of at least 5 independent experiments. Supporting Information, Fig. 7 Expression of PTK2 and SKP2 in human triple‐negative breast cancers (TNBC). Expression profiles were extracted from single cell RNA sequencing data [63] registered in the Broad Institute Single Cell Portal (https://singlecell.broadinstitute.org/single_cell). In (A), TNBC cell populations are depicted with different colors. Cells expressing high mRNA levels of PTK2 (B) or SKP2 (C) are shown as red or green dots, respectively. Co-expression is visualized in (D) according to the color intensities right panel. Note that expression of PTK2 is reversely correlated with that of SKP2 in certain TNBC cell populations, including malignant epithelial cells (inset). Supporting Information, Fig. 8 Expression of PTK2 and SKP2 expression in human colorectal carcinoma (CRC). Expression profiles were extracted from single cell RNA sequencing data [64] registered in the Broad Institute Single Cell Portal (https://singlecell.broadinstitute.org/single_cell). In (A), CRC cell populations are depicted with different colors. Cells expressing high mRNA levels of PTK2 (B) or SKP2 (C) are shown as red or light green dots, respectively. Co-expression is visualized in (D). Note that expression of PTK2 is reversely correlated with that of SKP2 in certain CRC cell populations, including malignant epithelial and stroma cells (inset) (PDF 572 kb)

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Vougioukalaki, M., Georgila, K., Athanasiadis, E.I. et al. Cell adhesion tunes inflammatory TPL2 kinase signal transduction. Cell. Mol. Life Sci. 79, 156 (2022). https://doi.org/10.1007/s00018-022-04130-7

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