PPM1D overexpression is associated with proinflammatory cytokine profile in response to NF-κB activation in human colorectal cancer cell line

Protein phosphatase PPM1D (Protein phosphatase, Mg²⁺/Mn²⁺ dependent, 1D) is one of the key regulators of DNA damage-induced stress response, cell cycle and apoptosis. PPM1D overexpression is detected in a large number of both solid (lung cancer, breast cancer, etc.) and hematological (acute myeloid leukemia) malignancies, making PPM1D an important prognostic marker in oncology. Special attention should be paid to PPM1D overexpression in colorectal cancer (CRC), the third most common oncological disease since this index correlates with increased tumor size, metastasis, unfavorable survival prognosis, and the development of drug resistance. Drug resistance may be associated with direct dephosphorylation of p53 and other components of the ATM-p53 signaling pathway by PPM1D, also acting as a negative regulator of NF- κB. It is likely that PPM1D overexpression in CRC tumor cells may lead to a decreased synthesis of important proinflammatory cytokines and development of an immunosuppressive tumor microenvironment, which may significantly worsen the outcomes of therapy. At present, the role of PPM1D in regulating the inflammatory response in CRC remains insufficiently studied. The aim of this work was to compare the effect of chemical PPM1D inhibition using its selective inhibitor GSK2830371 and genetic knockout of PPM1D in the human CRC cell line HT-29 under conditions of the NF-κB signaling pathway induction with TNF treatment. In this study, we used the HT-29 cell line, as well as earlier obtained HT-29 cell line with PPM1D gene knockout, and HT-29 cell line with PPM1D overexpression. The cell lines were cultured under standard conditions. For experiments, TNF (20 ng/mL) and GSK2830371 (10 μM) were supplied. The cell lines were incubated with GSK2830371 for 24 hours, and with TNF for 12 hours. Cytokine production was evaluated using xMAP INTELLIFLEX multiplex technology, and gene expression was measured by real-time PCR. Results: The multiplex assay of cytokine secretion levels revealed that, neither incubation of cells with GSK2830371 nor PPM1D knockout led to changes in the cytokine profile without stimulation as compared to the intact cell line, and did not cause a significant increase in production of pro-inflammatory cytokines and growth factors after TNF stimulation of the NF-κB pathway. In the case of the NF-κB pathway stimulation with TNF and PPM1D inhibition, we did not detect any significant increase in production of proinflammatory cytokines and growth factors. When evaluating the HT-29 cell line with PPM1D overexpression using real-time PCR, a statistically significant increase in TNF, IL-8, and IL-1b expression was observed in response to TNF treatment compared to the wild-type line. This finding is inconsistent with data on the negative regulation of NF-κB pathway by PPM1D phosphatase. In general, it was shown that PPM1D gene knockout didn’t significantly affect the level of cytokines (IL-8, GM-CSF, etc.) controlled by NF-κB transcription factor in the case of TNF induction, as compared with effects of a selective inhibitor GSK2830371. Meanwhile, overexpression of PPM1D was associated with increased expression of proinflammotory cytokine genes, e.g., IL-8, TNF, IL-1b, upon induction of the NF-κB pathway by TNF.