Tatiana S. Kalinina

Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of advanced non-small cell lung cancer (NSCLC). However, the limited predictive value of PD-L1 expression as a biomarker underscores the urgent need for more reliable predictors of ICI response. Interferon regulatory factor 1 (IRF1) is a transcription factor that lies downstream of interferon-γ signaling and directly regulates CD274 (PD-L1) transcription. Here, we performed a comprehensive bioinformatic analysis to identify microRNAs (miRNAs) that may be associated with IRF1 expression in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). Using data from The Cancer Genome Atlas (TCGA), we identified 20 miRNAs whose expression levels consistently and negatively correlated with IRF1 mRNA levels in both LUAD and LUSC. Among these, only hsa-miR-301b possesses conserved binding sites in the 3′UTR of IRF1 mRNA, suggesting direct post-transcriptional repression. For the remaining 19 miRNAs, we hypothesized an indirect mechanism of action. Further analysis revealed that hsa-miR-183 and hsa-miR-141 may target the transcription factor genes NFKB1 and STAT4, respectively, both of which positively correlate with IRF1 expression and are themselves associated with improved survival in ICI-treated patients. This study delineates a multi-layer miRNA regulatory network associated with IRF1 expression in NSCLC and identifies hsa-miR-301b, hsa-miR-183 and hsa-miR-141 as candidate upstream regulators of IRF1. Direct survival analysis for these miRNAs in ICI-treated cohorts was not feasible due to the lack of publicly available miRNA-seq data with treatment annotations; therefore, their clinical predictive value remains hypothetical, and experimental validation is required to assess their potential as predictors of ICI response.

Vitamin D deficiency is associated with the risk of atopic diseases and respiratory infections. The activated vitamin D receptor (VDR) forms a dimer with the retinoid X receptor alpha (RXRA) and binds to VDR/RXRA composite elements (CEs) in enhancers of target genes. However, VDR/RXRA CEs are identified in only 11.5% of cases in ChIP-Seq peaks. Our hypothesis was that VDR could form a VDR-Partner complex with transcription factor for which CEs have not yet been identified. We utilized Web-MCOT to search for novel VDR/Partner CEs in regulatory DNA. The potential formation of the VDR-Partner protein complex was assessed using the AlphaFold machine learning model. Through real-time RT-PCR, we measured the expression of immune system genes in a culture of U937 macrophage-like cells incubated with the active metabolite of vitamin D, calcitriol. We have predicted novel VDR/NR2C2 and VDR/PPARG CEs in the regulatory regions of immune system genes. We found potential synergism of VDR/NR2C2 and VDR/RXRA CEs in relation to the IRF5 gene, as well as potential synergism of VDR/PPARG and VDR/RXRA CEs for MAPK13. Predicting new regulatory relationships through the identification of new potential VDR/Partner CEs may provide insight into the deep mechanisms of vitamin D involvement in the pathogenesis of atopic dermatitis, bronchial asthma, allergic rhinitis, and pulmonary infections.

Tyrosine protein kinase HER2 plays an important role in carcinogenesis. In breast cancer (BC), the HER2 gene is amplified in approximately 20% of cases. Trastuzumab is used to treat BC with HER2 gene amplification. Trastuzumab is not effective in treating tumors with low HER2 expression, but trastuzumab deruxtecan was recently found to significantly improve prognosis in these patients. Nonetheless, there are still difficulties with accurate diagnosis of HER2-low BC, especially at the preoperative stage. In this study, when comparing the results between core needle biopsies and resection specimens using three immunohistochemistry scores (0, 1+, and 3+) of the HER2 protein level, we observed only moderate agreement (66.2%, κ = 0.486) in patients who did not undergo neoadjuvant therapy (n = 71). Other miRNA- or protein-coding genes regulated by HER2 signaling pathways may be additional markers for the scoring of the HER2 level. We measured levels of HER2-regulated miR-378a, -205, and -21 and mRNA levels of their target genes TRPS1, ITGA2, BCL6, and PTEN in BC surgical specimens. For estrogen receptor-positive BC (n = 64), we confirmed that the expression of miR-21, miR-378a, TRPS1, PTEN, and BCL6 is associated with the HER2 protein level. Moreover, the expression profile of miR-378a, BCL6, and PTEN differed between HER2 1+ and HER2 3+ tumors. TRPS1 expression was significantly higher in HER2 3+ tumors compared with HER2 0 tumors, but there was no difference in the amount of TRPS1 mRNA between HER2 1+ and HER2 3+ tumors. Thus, an analysis of the expression of miR-21, miR-378a, TRPS1, PTEN, and BCL6 may help to distinguish between HER2-negative, HER2-positive, and HER2-low BCs.

Smoking is an established risk factor for a variety of malignant tumors, the most well-known of which is lung cancer. Various molecular interactions are known to link tobacco smoke exposure to lung cancer, but new data are still emerging on the effects of smoking on lung cancer development, progression, and tumor response to therapy. In this study, we reveal in further detail the previously established association between smoking and hsa-mir-301a activity in lung squamous cell carcinoma, LUSC. Using different bioinformatic tools, we identified IRF1 as a key smoking-regulated target of hsa-mir-301a in LUSC. We further confirmed this relationship experimentally using clinical LUSC tissue samples and intact lung tissue samples. Thus, increased hsa-mir-301a levels, decreased IRF1 mRNA levels, and their negative correlation were shown in LUSC tumor samples. Additional bioinformatic investigation for potential pathways impacted by such a mechanism demonstrated IRF1’s multifaceted role in controlling the antitumor immune response in LUSC. IRF1 was then shown to affect tumor immune infiltration, the expression of immune checkpoint molecules, and the efficacy of immune checkpoint blockade therapy. As a result, here we suggest a smoking-regulated mir301a/IRF1 molecular axis that could modulate the antitumor immune response and immunotherapy efficacy in LUSC, opening up novel opportunities for future research.

Smoking is a risk factor for non-small cell lung cancer (NSCLC). The most common subtypes of NSCLC are lung adenocarcinoma (LAC) and squamous cell carcinoma (SCC). The cigarette smoke contains aryl hydrocarbon receptor (AhR) ligands, such as benzo(a)pyrene (BaP). By activating the AhR, BaP can change the expression of many genes, including miRNA-encoding genes. In this study, we have evaluated the expression of few miRNAs potentially regulated by AhR (miR-21, -342, -93, -181a, -146a), as well as CYP1A1, a known AhR target gene, in lung tumor samples from smoking (n=40) and non-smoking (n=30) patients with LAC and from smoking patients with SCC (n=40). We have also collected macroscopically normal lung tissue >5 cm from the tumor margin. We compared the obtained data on the miRNA expression in tumors with data from The Cancer Genome Atlas (TCGA). We found that in 76.7% of non-smoking LAC patients, CYP1A1 mRNA was not detected in tumor and normal lung tissues, while in smoking patients, CYP1A1 expression was detected in tumors in almost half of the cases (47.5% for SCC and 42.5% for LAC). The expression profile of AhR-regulated miRNAs differed between LAC and SCC and depended on the smoking status. In LAC patients, the expression of oncogenic miRNA-21 and miRNA-93 in tumors was higher than in normal lung tissue from the same patients. However, in SCC patients from our sample, the levels of these miRNAs in tumor and non-transformed lung tissue did not differ significantly. The results of our studies and TCGA data indicate that the expression levels of miRNA-181a and miRNA-146a in LAC are associated with smoking: expression of these miRNAs was significantly lower in tumors of smokers. It is possible that their expression is regulated by AhR and AhRR (AhR repressor), and inhibition of AhR by AhRR leads to a decrease in miRNA expression in tumors of smoking patients. Overall, these results confirm that smoking has an effect on the miRNA expression profile. This should be taken into account when searching for new diagnostic and therapeutic targets for NSCLC.

Introduction. Despite modern advances in research into the risk of development, immunological control and treatment options for lung cancer (LC), it is the leading cause of death from cancer. Tobacco smoking remains the predominant risk factor for the development of lung cancer, especially one of its aggressive subtypes, squamous cell lung cancer (SCLC). Benzo[a]pyrene, a component of cigarette smoke, promotes the activation of the aryl-hydrocarbon receptor (AhR). AhR regulates the expression of many oncogenes, including PD-L1, the positive status of which is an indication for immunotherapy, one of the main treatment strategies for SCLC. However, to improve the effectiveness of treatment of SCLC, further search for new diagnostic, prognostic and therapeutic markers is necessary. MicroRNAs (miRs), which are highly stable and present in biological fluids, can act as such markers.Objective. Search for microRNAs that could potentially serve as diagnostic markers or therapeutic targets for SCLC. For this purpose, microRNAs were selected whose promoter regions contain AhR binding sites or whose target is PD-L1.Materials and methods. A biocollection of tumor and conditionally normal lung tissue samples (n = 40) was collected at the thoracic department of the Novosibirsk Clinical Oncology Dispensary. The relative levels of selected miRNAs were examined using real-time reverse transcription-PCR (RT-PCR) technique.Results. The levels of miR-342 and miR-181a in SCLC tissues were reduced by 3 times relative to conditionally normal tissue. The expression of miR-181a and miR-155 is associated with tumor size (lower levels in tumors larger than 3 cm) and the presence of metastases in the lymph nodes (3- and 2-fold lower levels in cases with metastases). The level of miR-146a decreased by 3 times in patients with metastatic lesions of lymph nodes. A significant relationship between the levels of miR-93, miR-181a and miR-155 and the expression status of PD-L1 was also found.Conclusions. The expression profile of miR-146a, miR-93, miR-181a and miR-155 differs in SCLC patients depending on PD-L1 status and the presence or absence of lymph node metastases.

Aim. To assess the level of microRNA (miR) -21, -22, -126, -221 in patients with coronary artery disease (CAD) with borderline coronary artery stenosis depending on comorbidities and sex.Material and methods. We examined 37 patients with class 1-3 stable CAD aged 49-59 years with borderline (40-70%) coronary artery stenosis. The relative level of miRNA was determined using real-time polymerase chain reaction. Statistical analysis was performed using the non-parametric Mann-Whitney U-test. PResults. The miR-221 level was higher in the group of patients with stable CAD with borderline coronary artery stenosis with a metabolically unhealthy obesity (MUO) phenotype, but without diabetes (p=0,042). The level of miR-22 and miR-126 was higher in the group of patients with stable CAD phenotype with borderline stenosis and diabetes (p=0,007 and p=0,034, respectively). The analysis of miR levels in stable CAD patients depending on sex, without taking into account the phenotype, found that miR-21 and miR-221 values were higher in men (p=0,021 and p=0,014, respectively). The study of the sex characteristics of miR content in relation to different phenotypes revealed an increase of miR22 levels in men with MUO and diabetes (p=0,048) and an increase of miR-126 levels in women with concomitant diabetes in the comparison both with patients without MUO and diabetes (p=0,018), as well as with MUO and without diabetes (p=0,007).Conclusion. The study of the miRNA level in patients with CAD with borderline coronary artery stenosis is of great interest and reflects a promising direction in diagnosis based on comorbid pathology.

Many studies have shown that dichlorodiphenyltrichloroethane (DDT) exposure raises breast cancer risk. Another insecticide with similar properties is endosulfan, which has been actively used in agriculture after DDT prohibition. Previously, we have identified some estradiol-, progesterone-, and testosterone-sensitive microRNAs (miRNAs, miRs). Because DDT and endosulfan have estrogenic, antiandrogenic, and antiprogesterone properties, we hypothesized that these miRNAs are affected by the insecticides. We quantified relative levels of miRNAs and expression levels of their target genes in breast cancer MCF-7 cells treated with p,p′-DDT, o,p′-DDT, or endosulfan. We also quantified miR-19b expression, which, as previously shown, is regulated by estrogen. Here, we observed that miR-19b expression increased in response not only to estradiol but also to testosterone and progesterone. Treatment of MCF-7 cells with p,p′-DDT or endosulfan decreased the protein levels of apoptosis regulators TP53INP1 and APAF1. In cells treated with o,p′-DDT, the TP53INP1 amount decreased after 24 h of incubation, but increased after 48 h of incubation with insecticide. OXTR expression, which is known to be associated with breast carcinogenesis, significantly diminished under the exposure of all insecticides. In cells treated with p,p′-DDT or o,p′-DDT, the observed changes were accompanied by alterations of the levels of hormone-responsive miRNAs: miR-324, miR-190a, miR-190b, miR-27a, miR-193b, and miR-19b.

Despite the existing advances in the diagnosis and treatment of breast cancer (BC), the search for markers associated with the clinicopathological features of BC is still in demand. MiRNAs (miRs) have potential as markers, since a change in the miRNA expression profile accompanies the initiation and progression of malignant diseases. The receptors for estrogen, androgen, and progesterone (ER, AR, and PR) play an important role in breast carcinogenesis. Therefore, to search for miRNAs that may function as markers in BC, using bioinformatic analysis and the literature data, we selected 13 miRNAs whose promoter regions contain binding sites for ER or AR, or putative binding sites for ER, AR, and PR. We quantified their expression in MCF-7 cells treated with estradiol, progesterone, or testosterone. The levels of miRNAs sensitive to one or more of these hormones were quantified in BC samples (n = 196). We discovered that high expression levels of miR-190b in breast tumor tissue indicate a positive ER status, and miR-423 and miR-200b levels differ between patients with and without HER2 amplification. The miR-193b, -423, -190a, -324, and -200b levels were associated with tumor size or lymph node status in BC patients, but the presence of these associations depended on the status and expression level of ER, PR, HER2, and Ki-67. We also found that miR-21 expression depends on HER2 expression in ER- and/or PR-positive BC. The levels of miRNA were significantly different between HER2 0 and HER2 1+ tumors (p = 0.027), and between HER2 0 and HER2 2+, 3+ tumors (p = 0.005).

The oxytocin receptor (OXTR) plays an important role in childbirth, breastfeeding, and social interactions. Increasing evidence exists that OXTR is associated with the breast cancer (BC) initiation and progression. However, the mechanisms leading to its altered expression, the diagnostic or prognostic values of this receptor in BC are currently poorly understood. Here, we have evaluated the relative level of OXTR expression in BC samples (n = 107), and also investigated the effect of estradiol on its expression in MCF-7 and MDA-MB-231 cells. The level of OXTR expression was significantly lower in breast tumor tissue than in normal tissue obtained from the same patient. The OXTR expression depended on the status and expression of the estrogen receptor (ER): the level of OXTR mRNA was significantly lower in ER-negative BC samples compared to ER-positive BC samples. OXTR expression was also lower in samples from patients with luminal subtype with a low value of ER expression (0–5 score according to the IHC assay, Allred scoring) compared with samples with high ER expression (6–8 score). In luminal BC, OXTR expression was associated with the HER2 expression level: the OXTR mRNA level was higher in tumors with the HER2 IHC score of 1+ as compared to cases with the HER2 expression score of 2+, 3+. We also showed that estradiol increased the level of OXTR mRNA in MCF-7 cells, but not in ER-negative MDA-MB-231 cells. The data obtained indicate that changes in OXTR expression in BC tissues can be induced by increased ER expression. We found no association between OXTR and T or N stages and progesterone receptor expression.