Promotion effect of FOXCUT as a microRNA sponge for miR-24-3p on progression in triple-negative breast cancer through the p38 MAPK signaling pathway.

BACKGROUND: Triple-negative breast cancer (TNBC) is a type of highly invasive breast cancer with a poor prognosis. According to new research, long noncoding RNAs (lncRNAs) play a significant role in the progression of cancer. Although the role of lncRNAs in breast cancer has been well reported, few studies have focused on TNBC. This study aimed to explore the biological function and clinical significance of forkhead box C1 promoter upstream transcript (FOXCUT) in triple-negative breast cancer. METHODS: Based on a bioinformatic analysis of the cancer genome atlas (TCGA) database, we detected that the lncRNA FOXCUT was overexpressed in TNBC tissues, which was further validated in an external cohort of tissues from the General Surgery Department of the First Affiliated Hospital of Nanjing Medical University. The functions of FOXCUT in proliferation, migration, and invasion were detected in vitro or in vivo. Luciferase assays and RNA immunoprecipitation (RIP) were performed to reveal that FOXCUT acted as a competitive endogenous RNA (ceRNA) for the microRNA miR-24-3p and consequently inhibited the degradation of p38. RESULTS: lncRNA FOXCUT was markedly highly expressed in breast cancer, which was associated with poor prognosis in some cases. Knockdown of FOXCUT significantly inhibited cancer growth and metastasis in vitro or in vivo. Mechanistically, FOXCUT competitively bounded to miR-24-3p to prevent the degradation of p38, which might act as an oncogene in breast cancer. CONCLUSION: Collectively, this research revealed a novel FOXCUT/miR-24-3p/p38 axis that affected breast cancer progression and suggested that the lncRNA FOXCUT could be a diagnostic marker and therapeutic target for breast cancer.

Comprehensive evaluation of cell death-related genes as novel diagnostic biomarkers for breast cancer.

Background: Breast cancer (BRCA) ranks first among cancers in terms of incidence and mortality rates in women, primarily owing to metastasis, chemo-resistance, and heterogeneity. To predict long-term prognosis and design novel therapies for BRCA, more sensitive markers need to be explored. Methods: Data from 1089 BRCA patients were downloaded from TCGA database. Pearson's correlation analysis and univariate and multivariate Cox regression analyses were performed to assess the role of cell death-related genes (CDGs) in predicting BRCA prognosis. Kaplan-Meier survival curves were generated to compare the overall survival in the two subgroups. A nomogram was constructed using risk scores based on the five CDGs and other clinicopathological features. CCK-8, EdU incorporation, and colony formation assays were performed to verify the inhibitory effect of NFKBIA on BRCA cell proliferation. Transwell assay, flow cytometry, and immunohistochemistry analyses were performed to ascertain the biological function of NFKBIA. Results: Five differentially expressed CDGs were detected among 156 CDGs. The risk score for each patient was then calculated based on the expression levels of the five CDGs. Distinct differences in immune infiltration, expression of immune-oncological targets, mutation status, and half-maximal inhibitory concentration values of some targeted drugs were observed between the high- and low-risk groups. Finally, in vitro cell experiments verified that NFKBIA overexpression suppresses the proliferation and migration of BRCA cells. Conclusions: Our study revealed that some CDGs, especially NFKBIA, could serve as sensitive markers for predicting the prognosis of patients with BRCA and designing more personalized clinical therapies.

Deubiquitinase USP19 modulates apoptotic calcium release and endoplasmic reticulum stress by deubiquitinating BAG6 in triple negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC), a heterogeneous subtype of breast cancer (BC), had poor prognosis. Endoplasmic reticulum (ER) stress was responsible for cellular processes and played a crucial role in the cell function. ER stress is a complex and dynamic process that can induce abnormal apoptosis and death. However, the underlying mechanism of ER stress involved in TNBC is not well defined. METHODS: We identified ubiquitin-specific protease 19 (USP19) as a TNBC negative regulator for further investigation. The effects of USP19 on BC proliferation were assessed in vitro using proliferation test and cell-cycle assays, while the effects in vivo were examined using a mouse tumorigenicity model. Through in vitro flow cytometric analyses and in vivo TUNEL assays, cell apoptosis was assessed. Proteomics was used to examine the proteins that interact with USP19. RESULTS: Multiple in vitro and in vivo tests showed that USP19 decreases TNBC cell growth while increasing apoptosis. Then, we demonstrated that USP19 interacts with deubiquitinates and subsequently stabilises family molecular chaperone regulator 6 (BAG6). BAG6 can boost B-cell lymphoma 2 (BCL2) ubiquitination and degradation, thereby raising ER calcium (Ca2+ ) levels and causing ER stress. We also found that the N6 -methyladenosine (m6 A) "writer" methyltransferase-like 14 (METTL14) increased global m6 A modification. CONCLUSIONS: Our study reveals that USP19 elevates the intracellular Ca2+ concentration to alter ER stress via regulation of BAG6 and BCL2 stability and may be a viable therapeutic target for TNBC therapy.

LHX2 Is a Potential Biomarker and Associated with Immune Infiltration in Breast Cancer.

Worldwide, breast cancer is the most common malignancy. LHX2, a member of the LIM homeobox gene family and a transcription factor, plays a crucial role in numerous tumors, but the function of LHX2 in breast cancer progression remains unknown. In this study, we show that LHX2 is upregulated in breast cancer tissues and positively correlated with breast cancer progression. Meanwhile, the clinical characteristics of breast cancer and LHX2 expression showed a strong correlation. GSEA showed that a high LHX2 expression may activate the T-cell activation pathway, PI3K/AKT/mTOR signaling pathway, and apoptosis pathway. Moreover, ssGSEA showed that Th1 cells and Th2 cells had a positive correlation with LHX2 expression in breast cancer. Experiments showed that LHX2 promotes the proliferation, colony formation, migration, and invasion of breast cancer cells. Immunohistochemistry and immunofluorescence assays helped to analyze LHX2-associated immune infiltration in breast cancer. A Western blot assay proved that LHX2 activated the PI3K/AKT/mTOR pathway and the apoptosis pathway. A TUNEL assay confirmed that LHX2 inhibited apoptosis. Taken together, LHX2 plays a vital role in breast cancer's progression and prognosis and could be an immune infiltration biomarker for breast cancer, and LHX2 activates the PI3K/AKT/mTOR pathway and apoptosis pathway in breast cancer.

Integrated tumor genomic and immune microenvironment analysis identifies predictive biomarkers associated with the efficacy of neoadjuvant therapy for triple-negative breast cancer.

BACKGROUND: Although neoadjuvant chemotherapy (NAC) is currently the best therapy for triple-negative breast cancer (TNBC), resistance still occurs in a considerable proportion, thus it is crucial to understand resistance mechanisms and identify predictive biomarkers for patients selection. METHODS: Biopsy samples were collected from 21 patients with TNBC who underwent NAC. Whole-exome sequencing (WES), targeted sequencing, and multiplex immunohistochemistry (mIHC) were carried out on the clinical samples and used to identify and validate potential biomarkers associated with response to NAC. In addition, data on 190 TNBC patients who had undergone chemotherapy were obtained from The Cancer Genome Atlas (TCGA) and analyzed to further validate our findings. RESULTS: Both the tumor mutational burden (TMB) and tumor neoantigen burden (TNB) were significantly higher in responders than in non-responders. Higher response rates and longer survival rates were observed in patients with higher TMB. Patients with higher ratios of CD8 to M2 macrophages had higher response rates and improved survival rates. Finally, the integrated analysis demonstrated that the combination of TMB and the ratio of CD8 T cells to M2 macrophages could further distinguish patients who benefitted from the treatment in both enrolled patients and public data. CONCLUSIONS: The findings of this study indicated that the combination of TMB and the ratio of CD8 T cells to M2 macrophages may be a potential biomarker for improving the recognition of NAC responders, thereby providing a basis for developing precision NAC regimens.

Comprehensive analysis of pyroptotic gene prognostic signatures associated with tumor immune microenvironment and genomic mutation in breast cancer.

Background: Breast cancer is becoming a tumor with the highest morbidity rate, and inflammation-induced cell death namely pyroptosis reportedly plays dual roles in cancers. However, the specific mechanism between pyroptosis and the clinical prognosis of breast cancer patients is indistinct. Hence, novel pyroptosis-related biomarkers and their contributing factors deserve further exploration to predict the prognosis in breast cancer. Methods: Pearson's correlation analysis, and univariate and multivariate Cox regression analysis were utilized to obtain six optimal pyroptosis-related gene prognostic signatures (Pyro-GPS). The risk score of each breast cancer patient was calculated. Next, a Pyro-GPS risk model was constructed and verified in TCGA cohort (n=1,089) and GSE20711 cohort (n=88). Then analyses of immune microenvironment, genomic variation, functional enrichment, drug response and clinicopathologic feature stratification associated with the risk score of Pyro-GPS were performed. Subsequently, a nomogram based on the risk score and several significant clinicopathologic features was established. Ultimately, we further investigated the role of CCL5 in the biological behavior of MDA-MB-231 cell line. Results: The low-risk breast cancer patients have better survival outcomes than the high-risk patients. The low-risk patients also show higher immune cell infiltration levels and immune-oncology target expression levels. There is no significant difference in genetic variation between the two risk groups, but the frequency of gene mutations varies. Functional enrichment analysis shows that the low-risk patients are prominently correlated with immune-related pathways, whereas the high-risk patients are enriched in cell cycle, ubiquitination, mismatch repair, homologous recombination and biosynthesis-related pathways. Pyro-GPS is positively correlated with the IC50 of anti-tumor drugs. Furthermore, comprehensive analyses based on risk score and clinicopathological features were performed to predict the prognosis of breast cancer patients. Additionally, in vitro experiments confirmed that breast cancer cells with high expression of CCL5 had weaker proliferation, invasion and metastasis abilities as well as stronger apoptosis and cell cycle arrest abilities. Conclusions: The risk score of Pyro-GPS can serve as a promising hallmark to predict the prognosis of BRCA patients. Risk score evaluation may assist to acquire relevant information of tumor immune microenvironment, genomic mutation status, functional pathways and drug sensitivity, and thus provide more effective personalized strategies.

Comprehensive Analysis of the Immune-Oncology Targets and Immune Infiltrates of N 6-Methyladenosine-Related Long Noncoding RNA Regulators in Breast Cancer.

Breast cancer (BRCA) has become the highest incidence of cancer due to its heterogeneity. To predict the prognosis of BRCA patients, sensitive biomarkers deserve intensive investigation. Herein, we explored the role of N 6-methyladenosine-related long non-coding RNAs (m6A-related lncRNAs) as prognostic biomarkers in BRCA patients acquired from The Cancer Genome Atlas (TCGA; n = 1,089) dataset and RNA sequencing (RNA-seq) data (n = 196). Pearson's correlation analysis, and univariate and multivariate Cox regression were performed to select m6A-related lncRNAs associated with prognosis. Twelve lncRNAs were identified to construct an m6A-related lncRNA prognostic signature (m6A-LPS) in TCGA training (n = 545) and validation (n = 544) cohorts. Based on the 12 lncRNAs, risk scores were calculated. Then, patients were classified into low- and high-risk groups according to the median value of risk scores. Distinct immune cell infiltration was observed between the two groups. Patients with low-risk score had higher immune score and upregulated expressions of four immune-oncology targets (CTLA4, PDCD1, CD274, and CD19) than patients with high-risk score. On the contrary, the high-risk group was more correlated with overall gene mutations, Wnt/ß-catenin signaling, and JAK-STAT signaling pathways. In addition, the stratification analysis verified the ability of m6A-LPS to predict prognosis. Moreover, a nomogram (based on risk score, age, gender, stage, PAM50, T, M, and N stage) was established to evaluate the overall survival (OS) of BRCA patients. Thus, m6A-LPS could serve as a sensitive biomarker in predicting the prognosis of BRCA patients and could exert positive influence in personalized immunotherapy.

The Efficacy and Toxicity of Neoadjuvant Chemotherapy Regimens of Epirubicin Plus Cyclophosphamide Followed by Docetaxel or Paclitaxel in Female Breast Cancer Patients.

PURPOSE: To retrospectively analyze the efficacy and toxicity of epirubicin plus cyclophosphamide followed by docetaxel (EC-D) and epirubicin plus cyclophosphamide followed by paclitaxel (EC-P) efficacy as neoadjuvant chemotherapy regiments by pathological complete response (pCR) in this study. METHODS: In total, 455 patients diagnosed with breast cancer who received NAC from January 2014 to January 2019 were enrolled. Of which, 109 patients received EC-D (E: 90, C: 600, D: 80, all in mg/m2) and 346 were treated with EC-P (E: 90, C: 600, D: 175, all in mg/m2). Efficacy of NAC regimens was evaluated by pCR, and the toxicity was studied. Chi-squared test was used at p=0.05. RESULTS: In EC-D, 11 patients received ypT0/isN0, and 6 of them got ypT0N0. Analogously, 67 patients receiving received EC-P obtained ypT0/isN0, and 43 people of them acquired ypT0N0. The rate of pCR in EC-P was higher than EC-D. Patients with ER (-), PR (-), Her-2 (+) and high Ki-67 index were easier to were more likely to acquire pCR. Two pCRs were described, the pCR of NAC differed according to the definition. In terms of side effects, there was no significant difference in platelet and urea, but the decrease of hemoglobin and creatinine levels after EC-P treatment was more significant than that after EC-D treatment. CONCLUSION: The efficacy of EC-P is better than EC-D if pCR is to be determined as a surrogate end-point for prognosis. The patients with anemia or renal insufficiency who need to receive NAC should choose EC-D.

Identification of Recurrent Variants in BRCA1 and BRCA2 across Multiple Cancers in the Chinese Population.

BRCA1 and BRCA2 as important DNA repair genes have been thoroughly investigated in abundant studies. The potential relationships of BRCA1/2 pathogenic variants between multicancers have been verified in Caucasians but few in Chinese. In this study, we performed a two-stage study to screen BRCA1/2 pathogenic variants or variants of uncertain significance (VUS) with 7580 cancer cases and 4874 cancer-free controls, consisting of a discovery stage with 70 familial breast cancer cases and a subsequent validation stage with 7510 cases (3217 breast cancer, 1133 cervical cancer, 2044 hepatocellular carcinoma, and 1116 colorectal cancer). 48 variants were obtained from 70 familial breast cancer cases after BRCA1/2 exon detection, and finally, 20 pathogenic variants or VUS were selected for subsequent validation. Four recurrent variants in sporadic cases (BRCA1 c.4801A>T, BRCA1 c.3257del, BRCA1 c.440del, and BRCA2 c.7409dup) were identified and three of them were labeled Class 5 by ENIGMA. Two variants (BRCA1 c.3257del and c.440del) were specific in breast cancer cases, while BRCA2 c.7409dup and c.4307T>C were detected in two hepatocellular carcinoma patients and the BRCA1 c.4801A>T variant in one cervical cancer patient, respectively. Moreover, BRCA1 c.3257del was the most frequent variant observed in Chinese sporadic breast cancer and showed increased proliferation of BRCA1 c.3257del-overexpressing triple-negative breast cancer cell lines (MDA-MB-231) in vitro. In addition to the known founder deleterious mutations, our findings highlight that the recurrently pathogenic variants in breast cancer cases could be taken as candidate genetic screening loci for a more efficient genetic screening of the Chinese population.

Effects of Chemotherapy on Serum Lipids in Chinese Postoperative Breast Cancer Patients.

PURPOSE: Chemotherapy is a comprehensive therapy for breast cancer; nevertheless, its associated adverse effects are drawing increasing attention with the continuous improvement of the efficacy. The changes in serum lipids of breast cancer patients caused by chemotherapy have been reported by previous studies, whereby the former increase the incidence rate of cardiovascular disorders. However, the variations in the changes of serum lipids with different chemotherapy regimens have seldom been reported. METHODS: From January 2011 to December 2017, 1740 breast cancer patients treated with chemotherapy were recruited at the First Affiliated Hospital of Nanjing Medical University. The chemotherapy regimens included anthracycline-based, taxane-based, and anthracycline-plus-taxane-based regimens, dose-dense and standard-interval regimens. Lipid profiles that contained TG (triglyceride), TC (total cholesterol), HDL-C (high-density lipoprotein cholesterol), LDL-C (low-density lipoprotein cholesterol) and Lpa (lipoprotein a) levels were collected prior to the first, second and last cycles of chemotherapy. The changes of serum lipids with the same or different chemotherapy regimens were analyzed and compared. RESULTS: It was observed that the levels of TG, TC, LDL-C and Lpa increased significantly while that of HDL-C decreased after adjuvant chemotherapy in breast cancer patients (P<0.05). Besides, dose-dense regimens had more influence in TG and HDL-C and less influence in TC and LDL-C than standard-interval regimens. HDL-C was more sensitive to anthracycline-based regimens than taxane-based regimens. The level of TG with anthracycline-plus-taxane-based regimens was higher than that with only anthracycline-based or taxane-based regimens, and the level of HDL-C with anthracycline-plus-taxane-based regimen showed lower than that with taxane-based regimen. CONCLUSION: In summary, this study proposed that dyslipidemia was strongly associated with chemotherapy in Chinese breast cancer patients after operative treatment. Furthermore, the changes in levels of serum lipids varied among patients with different chemotherapy regimens and taxane had less effect on dyslipidemia than anthracycline.

Genetic variations within alternative splicing associated genes are associated with breast cancer susceptibility in Chinese women.

BACKGROUND: Alternative splicing regulates most of protein-coding genes by producing diverse messenger RNA transcripts; and mis-splicing events can induce aberrant protein isoforms that contribute to cancer development. It is possible that genetic variations in splicing associated genes may regulate the formation of transcripts and multiple protein isoforms by affecting the splice regulatory elements. In this study, we aimed to determine whether genetic variations in the crucial alternative-splicing genes were associated with breast cancer risk. MATERIALS AND METHODS: A case-control study was conducted with 1064 breast cancer cases and 1073 healthy controls from China. A total of 16 tagging polymorphisms within three splicing factor-associated genes (SFRS3, ESRP1 and ESRP2) were genotyped by using Infinium BeadChip. The association between the polymorphisms and risk of breast cancer was evaluated by computing odds ratios (OR) and 95% confidence intervals (CIs). RESULTS: The genotype distribution of rs2145048 in SFRS3 was different between cases and controls (Bonferroni corrected P = 0.022). After adjusting for age, age at menarche and menopausal status, the A allele of rs2145048 showed an inverse association with breast cancer risk in the additive model (adjusted OR = 0.81, 95% CI = 0.71-0.92, P = 0.001, Bonferroni corrected P = 0.016). In the stratification analysis, the association between rs2145048 A allele and breast cancer remained significant in subgroups of earlier menarche, older first born, premenopausal status, and ER/PR negative status. CONCLUSIONS: This study provided the first evidence that SFRS3 rs2145048 was associated with breast cancer susceptibility in Chinese women, which might represent a biomarker to improve the identification of individuals at high risk of this malignancy.

Peripheral inflammation/immune indicators of chemosensitivity and prognosis in breast cancer patients treated with neoadjuvant chemotherapy.

INTRODUCTION: Neoadjuvant chemotherapy (NAC) has become a standard treatment for locally advanced breast cancer. The present study was designed to investigate the predictive value of different peripheral inflammation/immune biomarker responses to NAC and prognosis in breast cancer patients. MATERIALS AND METHODS: A total of 180 breast cancer patients treated with NAC in the First Affiliated Hospital with Nanjing Medical University between January 2008 and March 2015 were enrolled in the study. The associations between inflammation/immune indicators and pathological complete response (pCR) were determined, and the prognostic value of inflammation/immune indicators was also evaluated. RESULTS: In the univariate analysis, patients with a high pretreatment peripheral lymphocyte count (.2.06×109/L) showed a higher pCR rate than those with a low lymphocyte count (23.9% vs 10.4%, P=0.023). The pCR rate of patients with a neutrophil: lymphocyte ratio ≤2.15 was significantly higher than that of patients with a high neutrophil: lymphocyte ratio (20% vs 7.8%; P=0.03). However, multivariate analysis revealed that only the high lymphocyte count was predictive for pCR (odds ratio: 4.375, 95% CI: 1.429-13.392, P=0.010). In the survival analysis, patients with a higher neutrophil count (.2.65×109/L) were confirmed to have a shorter disease-free survival (hazard ratio: 4.322, 95% CI: 1.028-18.174, P=0.046), and the high neutrophil count was significantly associated with lymphovascular invasion (P=0.037). CONCLUSION: We demonstrated that a high level of baseline peripheral lymphocyte count can be a predictor for high efficacy of NAC for breast cancer patients, and low baseline peripheral neutrophil count may contribute to the favorable disease-free survival.

Mannitol improves cerebral oxygen content and postoperative recovery after prolonged retroperitoneal laparoscopy.

BACKGROUND: Prolonged pneumoperitoneum has cerebral adverse effects that may delay recovery and cause postoperative cognitive changes. The purpose of this study was to investigate the effect of mannitol infusion after pneumoperitoneum initiation on cerebral oxygen balance and quality of postoperative recovery in patients undergoing prolonged retroperitoneal laparoscopy. METHODS: Forty patients scheduled for retroperitoneal laparoscopic radical excision of prostatic carcinoma were randomly divided into two groups (n = 20, each) to receive either 0.5 g/kg of 20% mannitol 150 min after the initiation of pneumoperitoneum or an equal volume of 0.9% normal saline. After surgery, time to extubation and recovery time were recorded. The Observer's Assessment of Alertness/Sedation (OAA/S) scale was used to assess the quality of recovery. The Mini-Mental State Exam (MMSE) was given to test cognitive function preoperatively and at 1, 2, and 3 h after extubation. Blood samples from the jugular bulb and the radial artery were collected for blood gas analysis before CO2 insufflation and at 10, 60, and 180 min after insufflation. RESULTS: In the control group (without mannitol), the difference between arterial and venous oxygen content (CaO2-CvO2) before insufflation (6.21 ± 2.58 mL/dL) was significantly greater than it was 3 h after insufflation (2.63 ± 1.29 mL/dL; p < 0.05). Furthermore, 3 h after insufflation, the CaO2-CvO2 also was higher in the group that had been administered mannitol (5.93 ± 1.98 mL/dL) than it was in the control group at that time (p < 0.05). Lactic acid in both arterial and jugular venous blood of the control group at 3 h postinsufflation (2.39 ± 0.89 and 2.51 ± 0.72 mg/dL, respectively) had increased significantly from the preinsufflation values (1.18 ± 0.82 and 1.1 ± 0.85 mg/dL). In the group that received mannitol, the lactic acid levels 3 h postinsufflation were essentially the same as the preinsufflation values. The recovery and extubation times in those receiving mannitol (12.19 ± 2.12 and 20.14 ± 3.62 min, respectively) were significantly shorter than in the control group (21.25 ± 3.61 and 28.79 ± 4.73 min; p < 0.05). The OAAS scores of the mannitol group at the time of extubation and 10 min afterward was significantly higher than these scores in the control group (p < 0.05). One hour and 2 h after extubation, the cognitive function score of the mannitol group was significantly higher than for the control group (p < 0.05). CONCLUSIONS: After prolonged retroperitoneal laparoscopy, there is an imbalance between oxygen supply and demand. A small dose of mannitol can effectively improve cerebral oxygen metabolism, recovery, and cognitive function after the operation.