Non-small cell lung cancer (NSCLC) has limited treatment options. Expression of the RNA-binding protein (RBP) Musashi-2 (MSI2) is elevated in a subset of non-small cell lung cancer (NSCLC) tumors upon progression, and drives NSCLC metastasis. We evaluated the mechanism of MSI2 action in NSCLC to gain therapeutically useful insights. Reverse phase protein array (RPPA) analysis of MSI2-depleted versus control KrasLA1/+; Trp53R172HΔG/+ NSCLC cell lines identified EGFR as a MSI2-regulated protein. MSI2 control of EGFR expression and activity in an NSCLC cell line panel was studied using RT-PCR, Western blots, and RNA immunoprecipitation. Functional consequences of MSI2 depletion were explored for cell growth and response to EGFR-targeting drugs, in vitro and in vivo. Expression relationships were validated using human tissue microarrays. MSI2 depletion significantly reduced EGFR protein expression, phosphorylation, or both. Comparison of protein and mRNA expression indicated a post-transcriptional activity of MSI2 in control of steady state levels of EGFR. RNA immunoprecipitation analysis demonstrated that MSI2 directly binds to EGFR mRNA, and sequence analysis predicted MSI2 binding sites in the murine and human EGFR mRNAs. MSI2 depletion selectively impaired cell proliferation in NSCLC cell lines with activating mutations of EGFR (EGFRmut). Further, depletion of MSI2 in combination with EGFR inhibitors such as erlotinib, afatinib, and osimertinib selectively reduced the growth of EGFRmut NSCLC cells and xenografts. EGFR and MSI2 were significantly co-expressed in EGFRmut human NSCLCs. These results define MSI2 as a direct regulator of EGFR protein expression, and suggest inhibition of MSI2 could be of clinical value in EGFRmut NSCLC.
Purpose of the study. An analysis of indices of free radical oxidation and respiration of mitochondria of heart cells in a malignant process in presence of diabetes mellitus and chronic neurogenic pain in experimental animals.Materials and methods. The study included outbred female rats (n=32) and С57ВL/6 female mice (n=84). Experimental groups of rats were: intact group 1 (n=8), control group 1 (n=8) with diabetes mellitus (DM), comparison group 1 (n=8) with standard subcutaneous transplantation of Guerin’s carcinoma, main group 1 (n=8) with Guerin’s carcinoma transplanted after 1 week of persistent hyperglycemia. Experimental groups of mice were: intact group 2 (n=21), control group 2 (n=21) with a model of chronic neurogenic pain (CNP), comparison group 2 (n=21) with standard subcutaneous transplantation of melanoma (B16/F10), main group 2 (n=21) (CNP+B16/F10) with melanoma transplanted 3 weeks after the CNP model creation. Heart mitochondria were isolated by differential centrifugation. Levels of cytochrome C (ng/mg of protein), 8-hydroxy-2'-deoxyguanosine (8-OHdG) (ng/mg of protein), and malondialdehyde (MDA) (μmol/g of protein) were measured in mitochondrial samples by ELISA. Statistical analysis was performed using the Statistica 10.0 program.Results. DM in rats upregulated 8-OHdG by 6.3 times and MDA by 1.9 times (р=0.0000) and downregulated cytochrome C by 1.5 times (р=0.0053) in heart cell mitochondria, compared to intact values. DM+Guerin’s carcinoma in rats increased 8-OHdG by 14.0 times and MDA by 1.7 times (р=0.0000) and decreased cytochrome C by 1.5 times (р=0.0000), compared to intact values. CNP in mice did not affect the studied parameters in mitochondria of the heart. CNP+B16/F10 in mice increased 8-OHdG by 7.1 times and MDA by 1.6 times (р=0.0000) and decreased cytochrome C by 1.6 times (р=0.0008).Conclusions. Comorbidity (diabetes mellitus, chronic neurogenic pain) together with malignant pathology aggravates mitochondrial dysfunction of heart cells with destabilization of the respiratory chain mediated by free radical oxidation processes.
Background The RNA-binding protein Musashi-2 (MSI2) controls the translation of proteins that support stem cell identity and lineage determination and is associated with progression in some cancers. We assessed MSI2 as potential clinical biomarker in colorectal cancer (CRC) and tubulovillous adenoma (TA) of colon mucosa. Methods We assessed 125 patients, of whom 20 had polyps of the colon (TAs), and 105 had CRC. Among 105 patients with CRC, 45 had stages I-III; among metastatic CRC (mCRC) patients, 31 had synchronous and 29 metachronous liver metastases. We used immunohistochemistry to measure MSI2 expression in matching specimens of normal tissue versus TAs, primary CRC tumors, and metastases, correlating expression to clinical outcomes. We analyzed the biological effects of depleting MSI2 expression in human CRC cells. Results MSI2 expression was significantly elevated in polyps versus primary tissue, and further significantly elevated in primary tumors and metastases. MSI2 expression correlated with decreased progression free survival (PFS) and overall survival (OS), higher tumor grade, and right-side localization (p = 0.004) of tumors. In metastases, high MSI2 expression correlated with E-cadherin expression. Knockdown of MSI2 in CRC cells suppressed proliferation, survival and clonogenic capacity, and decreased expression of TGFβ1, E-cadherin, and ZO1. Conclusion Elevated expression of MSI2 is associated with pre-cancerous TAs in the colonic mucosa, suggesting it is an early event in transformation. MSI2 expression is further elevated during CRC progression, and associated with poor prognosis. Depletion of MSI2 reduces CRC cell growth. These data imply a causative role of MSI2 overexpression at multiple stages of CRC formation and progression.
Background: Musashi-2 (MSI2) is a member of RNA-binding protein family that regulates mRNA translation of numerous intracellular targets and influences maintenance of stem cell identity. This study assessed MSI2 as a potential clinical biomarker in non-small cell lung cancer (NSCLC). Methods: The current study included 40 patients with NSCLC, of whom one presented with stage 1, 14 presented with stage II, 15 presented with stage III, and 10 patients had stage IV. All patients received standard of care treatments. All patient samples were obtained before treatment started. We used immunohistochemical (IHC) approach to measure MSI2 protein expression in matching specimens of normal lung versus tumor tissues, and primary versus metastatic tumors, followed by correlative analysis in relation to clinical outcomes. In parallel, clinical correlative analysis of MSI2 mRNA expression was performed in silico using publicly available datasets (TCGA/ICGC and KM plots). Results: MSI2 protein expression in patient samples was significantly elevated in NSCLC primary tumors versus normal lung tissue (P=0.03). MSI2 elevated expression positively correlated with a decreased progression free survival (PFS) (P=0.026) combined for all stages and with overall survival (OS) at stage IV (P=0.013). Elevated MSI2 expression on RNA level was confirmed in primary tumor versus normal tissue samples in TCGA dataset (P<0.0001), and positively correlated with decreased OS (P=0.02). No correlation was observed between MSI2 expression and age, sex, smoking, and treatment type. Conclusions: Elevated MSI2 expression in primary NSCLC tumors is associated with poor prognosis and can be used as a novel potential prognostic biomarker in NSCLC patients. Future studies in an extended patient cohort are warranted.
To study the effect of comorbid pathology: obesity of degree 2-3 on the level of sex steroid hormones and their receptors in the tumor and its surrounding tissue in patients with endometrial cancer (EC). Materials and methods. In 30 patients with endometrioid adenocarcinoma T1-3N0-1M0 (the main group, 15 females with obesity grade 2-3 (BMI≥35); the reference group 15 females with normal BMI) in samples of the tumor and its perifocal zone taken after surgical treatment, the levels of estradiol (E2), estrone (E1), testosterone (T), progesterone (P4), androgen receptors (AR), progesterone receptors (RP4), estrogen receptors (ERα and ERβ) were determined by ELISA method. Statistical analysis was performed with STATISTICA 10.0. Results. Obese EC patients showed longer healing of postoperative wounds, slow recovery, and more frequent tumor metastasizing to regional lymph nodes. In the tumor samples in all patients, compared with the intact endometrium, the levels of estrogens, testosterone and their receptors were higher. Obesity accompanying the malignant process led to a local increase in the levels of estrogens, testosterone, progesterone and AR, ERα and ERβ in the tumor. In the tumor samples, there were no significant differences from the presence of obesity in the levels of RP4. In the perifocal zone of the tumor in patients with comorbid pathology, compared with the parameters in the reference group, the level of E2, P4 and T was also higher, but the content of all steroid receptors was lower. Conclusion. Obesity aggravates hyperestrogenism and progesterone deficiency in adenocarcinoma and increases its enrichment with the androgen and estrogen receptors with the prevalence of ERα over ERβ that may cause the autocrine-paracrine regulation of the growth and metastasizing of the malignant process in patients with endometrial cancer.
Purpose of the study. Diabetes mellitus (DM) is considered an independent risk factor for higher cancer incidence and death rates. The system of insulin-like growth factors and their carrier proteins (IGF and IGFBP) and hyperglycemia create favorable conditions for the proliferation and metastasis of cancer cells.Materials and methods. Outbred male and female rats were divided into groups (n = 8 each): controls - with Guerin's carcinoma; main group - Guerin's carcinoma growing in presence of DM. Experimental DM was reproduces in animals by the single intraperitoneal alloxan injection (150 mg/kg body weight). After 10 days of the carcinoma growth, levels of IGF and IGFBP in the tumor and in it's perifocal area were measured using ELISA.Results. DM in females upregulated levels of glucose both in the tumor and in perifocal tissues by 1.8 (p < 0.05) and 8.1 times, respectively, but caused opposite changes in IGF-I - it's increase by 6.3 times in the tumor and decrease by 3.2 times in the perifocal area; as a result, such tumors with small primary nodes were more "aggressive" and actively metastasized. In males, induced DM downregulated levels of glucose, IGF-II and IGFBP2 in the carcinoma by 8.4, 3.1 and 1.7 (p < 0.05) times, respectively, and increased levels of IGF-I and IGFBP2 by 1.4 and 1.3 times (p < 0.05) in the perifocal area without changing glucose levels; as a result, tumor volumes exceeded the values in the standard growth, without metastasizing into visceral organs.Conclusion. We revealed gender differences in changing levels of glucose and IGF both in the tumor and in it's perifocal tissue in rats with Guerin's carcinoma growing in presence of DM; these differences could determine different tumor growth dynamics in male and female rats.
The aim is to study some mechanisms of regulation of apoptosis and self-organization in the mitochondria in the heart cells in female mice during the growth of experimental melanoma B16/ F10 linked with chronic neurogenic pain as comorbid pathology.
The aim is to evaluate the physiological parameters of the efficacy of cardiac mitochondria transplantation in male mice with chronic neurogenic pain and B16/F10 melanoma growth. Materials and methods. Male mice (n=37) of the C57BL/6 line were used in the research work. The animals covered experimental groups as follows: mice with chronic neurogenic pain (CNP) + B16/F10 melanoma (n=27); mice with CNB + B16/ F10 melanoma + mitochondrial therapy (MC therapy) (n=10). Mitochondria were isolated from the heart of an intact rat with the use of differential centrifugation. An introduction of mitochondria to mice was carried out daily intraperitoneally at a dose of 3.3 mg of protein for 3 weeks. Statistical analysis of the results is carried out with the Statistica 10.0 software. Results. On day 21 (week 3) of the experiment, macroscopically in the melanoma tissue in the group of animals with MC therapy, there were 2.5 times more necrosis cases than in the group without MC therapy. During the examination of the internal organs, no metastases were detected in the animals treated with MC therapy, while in 100% of the animals without MC therapy metastases were found in the lungs and in 95% of them in the spleen. In the animals received MC therapy, there was no damage to the heart muscle in 75% of the cases, while in the group of the animals without MC therapy, the presence of lesions in the form of bruises on the surface of the heart was macroscopically detected in 100% of the animals. Conclusion. Thus, intraperitoneal transplantation of intact heart mitochondria contributed to the prevention of myocardial infarction and metastases to internal organs in the C57BL/6 male mice with B16/F10 melanoma growing against the background of chronic neurogenic pain.
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