Tristetraprolin (tristetraprolin, TTP, ZFP36, encoded by the ZFP36 gene) is an RNA-binding protein (RBP) involved in post-transcriptional regulation of gene expression via degradation of target mRNAs. It is considered one of the key regulators of many cellular processes associated with malignancy. Approximately 15% of mRNAs contain AU-rich elements (AREs) in their 3'-untranslated regions (3'-UTRs) and may be regulated by RNA-binding proteins, one of which is TTP. It has been shown that TTP expression is significantly dysregulated in various types of cancer, including breast cancer (BC), and its involvement in the degradation of mRNAs associated with migration and invasion has been demonstrated. However, there is insufficient data on the impact of TTP on the expression of cytoskeleton-associated genes.
The study aimed to investigate the effects of TTP overexpression on the expression of cytoskeleton-associated genes SH3PXD2A, SH3PXD2B, CTTN, WIPF1, and WASL in the context of cell motility and invasion in a triple-negative breast cancer (TNBC) model, investigate the effects of doxorubicin on the expression of these genes and cell mobility, and evaluate the potential use of TTP as a biomarker in BC.
In this study, 49 transcript variants of genes involved in migration and invasion were analyzed in silico, and it was found that 24 of them contain AREs in their 3'-UTRs. Additionally, in silico analysis of the probability of TTP binding to mRNAs of SH3PXD2A, SH3PXD2B, CTTN, WIPF1, and WASL revealed a high probability of binding. To investigate the effect of TTP on the expression of target genes, a cell line MDA-MB-231, representing a highly invasive triple-negative breast cancer subtype (TNBC), with constitutive overexpression of ZFP36 was generated. Real-time quantitative PCR revealed that ZFP36 overexpression reduced SH3PXD2A and CTTN mRNA levels, while SH3PXD2B mRNA levels increased.
Further investigation via confocal microscopy revealed that MDA-MB-231 cells with TTP overexpression had significantly reduced cell areas compared to wild-type cells, but their shape remained unchanged. Additionally, the cells exhibited shorter and wider actin filaments as well as increased cortical actin intensity, suggesting a dysregulation in filament organization. Moreover, time-lapse live-cell imaging revealed that modified cells had decreased motility and directed movement capacity and demonstrated reduced invasive potential. Therefore, the data presented support existing research and expand current knowledge about potential direct or indirect roles of TTP in inhibiting metastasis.
Analysis of breast cancer tissue samples of different types revealed that ZFP36 expression was significantly higher in tumors of all types compared to adjacent tissues. Notably, ZFP36 levels were significantly higher in HER2-enriched tumors compared to other types. Additionally, the median survival in a cohort of patients with luminal B subtype breast cancer was significantly higher in the group with high ZFP36 expression, while an opposite correlation was observed in the cohort of HER+ patients. The data obtained in this study indicate that TTP cannot be used as a purely positive prognostic marker for breast cancer patients, at least for those with TNBC, as its high levels correlate with both favorable and unfavorable prognoses.
The study also demonstrated the possibility of inducing ZFP36 expression with clinically relevant concentrations of doxorubicin (DXR) (0.1, 0.5, and 1.0 µM). It was shown that DXR induces ZFP36 expression in a dose-dependent manner and dysregulates the expression of target cytoskeleton-associated genes. In the MCF7 cell line, the maximum level of TTP was found to be twice as high as in intact cells, while in the MDA-MB-231 cell line, it was 15 times higher. Whether such a sharp and significant increase in TTP levels occurs in vivo remains unclear, and further research is needed to investigate this effect in clinical settings. Additionally, it was shown that DXR decreases the motility of MDA-MB-231 cells in a dose-dependent manner and causes significant changes in their area, shape, and actin filament organization. The data obtained may limit the use of TTP as a purely positive prognostic marker for BC patients, particularly those with the TNBC, and expand current understanding of the effects of doxorubicin on the cytoskeleton.
The presented results expand the understanding of the impact of TTP on the invasive potential of triple-negative breast cancer cells, uncover new potential aspects of its role as a regulator of morphology and motility in these cells, describe changes in the expression of cytoskeleton-associated genes SH3PXD2A, SH3PXD2B, and CTTN under the influence of constitutive TTP overexpression in TNBC cells and DXR treatment, and highlight the controversial application of TTP as a purely positive prognostic biomarker in breast cancer.
