Artur Zajkowicz

We studied the associations between 3′UTR genetic variants in ADME genes, clinical factors, and the risk of breast cancer chemotherapy toxicity. Those variants and factors were tested in relation to seven symptoms belonging to myelotoxicity (anemia, leukopenia, neutropenia), gastrointestinal side effects (vomiting, nausea), nephrotoxicity, and hepatotoxicity, occurring in overall, early, or recurrent settings. The cumulative risk of overall symptoms of anemia was connected with AKR1C3 rs3209896 AG, ERCC1 rs3212986 GT, and >6 cycles of chemotherapy; leukopenia was determined by ABCC1 rs129081 allele G and DPYD rs291593 allele T; neutropenia risk was correlated with accumulation of genetic variants of DPYD rs291583 allele G, ABCB1 rs17064 AT, and positive HER2 status. Risk of nephrotoxicity was determined by homozygote DPYD rs291593, homozygote AKR1C3 rs3209896, postmenopausal age, and negative ER status. Increased risk of hepatotoxicity was connected with NR1/2 rs3732359 allele G, postmenopausal age, and with present metastases. The risk of nausea and vomiting was linked to several genetic factors and premenopausal age. We concluded that chemotherapy tolerance emerges from the simultaneous interaction of many genetic and clinical factors.

Background: PPM1D (WIP1) negatively regulates by dephosphorylation many proteins including p53 tumour suppressor. The truncating mutations (nonsense and frameshift) in exon 6 of PPM1D were found recently in blood cells of patients with breast, ovarian or colorectal cancer. These mutants code for gain-of-function PPM1D with retained phosphatase activity. Their significance in carcinogenesis is unknown. Methods: The exon 6 of PPM1D was sequenced in blood DNA of 543 non-small-cell lung cancer patients (NSCLC). The functional significance of selected PPM1D alterations (Arg458X, Lys469Glu) was compared with the wild-type gene and examined by recombinant DNA techniques, immunoblotting and luciferase reporter assays. Results: The frameshift mutations were found in five NSCLC patients (5/543; 0.92%), all of them had squamous cell carcinomas (5/328; 1.5%). All patients with the mutations were exposed, before the blood collection, to the DNA damaging agents as a part of chemotherapeutic regimen. Functional tests demonstrated that truncating mutation Arg458X causes enhancement of dephosphorylation activity of PPM1D toward serine 15 of p53, whereas Lys469Glu version is equivalent to the wild-type. Neither version of PPM1D (wild-type, Arg458X, Lys469Glu) significantly modulated the ability of p53 to transactivate promoters of the examined p53-target genes (BAX and MDM2). Conclusions: The truncating mutations of PPM1D are present in blood DNA of NSCLC patients at frequency similar to percentage determined for ovarian cancer patients. Our findings raise a question if the detected lesions are a result of chemotherapy.

Resveratrol is a natural compound that has been intensely studied due to its role in cancer prevention and potential as an anti-cancer therapy. Its effects include induction of apoptosis and senescence-like growth inhibition. Here, we report that two cancer cell lines (U-2 OS and A549) differ significantly in their molecular responses to resveratrol. Specifically, in U-2 OS cells, the activation of the p53 pathway is attenuated when compared to the activation in A549 cells. This attenuation is accompanied by a point mutation (458: CGA→TGA) in the PPM1D gene and overexpression of the encoded protein, which is a negative regulator of p53. Experimentally induced knockdown of PPM1D in U-2 OS cells resulted in slightly increased activation of the p53 pathway, most clearly visible as stronger phosphorylation of p53 Ser³⁷. When treated with nutlin-3a, a non-genotoxic activator of p53, U-2 OS and A549 cells both responded with substantial activation of the p53 pathway. Nutlin-3a improved the clonogenic survival of both cell lines treated with resveratrol. This improvement was associated with lower activation of DNA-damage signaling (phosphorylation of ATM, CHK2, and histone H2AX) and higher accumulation of cells in the G1 phase of the cell cycle. Thus, the hyperactivation of p53 by nutlin-3a helps to preserve the replicative potential of cells exposed to resveratrol.