PPM1D OVEREXPRESSION LEADS TO A PROINFLAMMATORY CYTOKINE PROFILE IN RESPONSE TO NF-κB ACTIVATION IN A HUMAN COLORECTAL CANCER CELL LINE

Abstract

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, because this indicator correlates with increased tumor size, metastasis, unfavorable survival prognosis, and the development of drug resistance. Drug resistance may be associated with the fact that PPM1D directly dephosphorylates p53 and other components of the ATM-p53 signaling pathway, and also acts as a negative regulator of NF-κB. It is likely that PPM1D overexpression in CRC tumor cells may lead to a decrease in the synthesis of important proinflammatory cytokines and the development of an immunosuppressive tumor microenvironment, which can significantly worsen the outcome of therapy. At present, the role of PPM1D in regulating the inflammatory response in CRC remains insufficiently studied. The aim of this study is to compare the effect of chemical inhibition of PPM1D using its selective inhibitor GSK2830371 and genetic knockout of PPM1D on the human colorectal cancer cell line HT-29 under conditions of NF-κB signaling pathway induction with TNF treatment.

In this work, the human colorectal cancer cell line HT-29 was used, as well as previously obtained HT-29 cell line with PPM1D gene knockout and HT-29 cell line with PPM1D overexpression. Cell lines were cultured under standard conditions. For experiments, TNF at a concentration of 20 ng/ml and GSK2830371 at a concentration of 10 μM were used. 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.

The multiplex assessment of cytokine secretion levels revealed that neither the incubation of cells with GSK2830371 nor PPM1D knockout led to changes in the cytokine profile without stimulation compared to the intact cell line, and did not cause a significant increase in the production of pro-inflammatory cytokines and growth factors in the case of NF-κB pathway stimulation using TNF. In the case of NF-κB pathway simulation with TNF and PPM1D inhibition, no significant increase in the production of proinflammatory cytokines and growth factors was detected. When evaluating the HT-29 cell line with PPM1D overexpression using real-time PCR, a statistically significant increase in the expression levels of TNF, IL-8, and IL-1b was observed in response to TNF treatment compared to the wild-type line. This finding is inconsistent with data on the negative regulation of the NF-κB pathway by PPM1D phosphatase.

As a result of the work, it was shown that PPM1D gene knockout didn’t significantly affect the level of cytikines (IL-8, GM-CSF, etc.) controlled by the trascription factor NF-κB in the case of TNF induction, compared with the use of the selective inhibitor GSK2833071. At the same time, overexpression of PPM1D led to an increase in the expression of proinflammotory cytokine genes, such as IL-8, TNF, IL-1b, upon induction of the NF-κB pathway by TNF.