miR-33a-5p in small extracellular vesicles as non-invasive biomarker for oxaliplatin sensitivity in human colorectal cancer cells

microRNAs (miRNAs) contained in small extracellular vesicles (sEVs) are candidates for non-invasive biomarkers. Oxaliplatin (L-OHP) has been approved for advanced colorectal cancer (CRC) chemotherapy. However, the response to L-OHP differs among CRC patients. In addition, CRC cells often acquire the resistance to L-OHP. This study aimed at the prediction of L-OHP sensitivity by measuring extracellular miRNAs levels. Firstly, we compared intracellular miRNAs expressions in L-OHP-sensitive CRC cells (SW620 and HCT116 cells) with those in acquired and intrinsic L-OHP-resistant cells. In microarray and real-time RT-PCR analyses, the intracellular miR-33a-5p, miR-210–3p, and miR-224–5p expressions were lower in acquired and intrinsic L-OHP-resistant CRC cells than sensitive cells. Furthermore, in SW620 cells, L-OHP sensitivity was decreased by miR-33a-5p inhibitor. On the other hand, miR-210–3p or miR-224–5p inhibitor did not affect L-OHP sensitivity in SW620 cells. Secondly, the amount of miR-33a-5p, miR-210–3p, and miR-224–5p in sEVs was compared. The amount of miR-33a-5p and miR-210–3p in sEVs secreted from acquired and intrinsic L-OHP-resistant cells tended to be small. miR-224–5p was not detected in sEVs secreted from three types of CRC cells examined. To the best of our knowledge, this is the first study demonstrating that miR-33a-5p and/or miR-210–3p in sEVs would be candidates for biomarkers of L-OHP sensitivity. In particular, miR-33a-5p is a promising candidate because it would be directly involved in L-OHP sensitivity.     

丝氨酸蛋白酶抑制剂(SERPIN)家族相关基因在结肠腺癌中预后模型的建立及应用

应用生物信息学方法,构建结肠腺癌(COAD)丝氨酸蛋白酶抑制剂(SERPIN)家族相关基因预后模型。从TCGA数据库和GEO数据库下载结肠腺癌(COAD)转录组和临床数据,根据数据中SERPINs家族基因的表达量对COAD患者进行一致性聚类分析;将数据随机均分为训练集(Train)组和验证集(Test)组,基于两个亚型的差异基因,利用Train组进行COX回归和Lasso回归构建预后模型,根据模型风险评分中位值将样本分为高、低风险两组,绘制高低风险组患者生存曲线;通过ROC曲线评价模型预测能力;利用Test组数据验证模型;构建列线图,评估患者生存率模型预测值与实际值的一致性;并利用利用ESTIMATE算法和CIBERSORT算法评估风险评分和肿瘤微环境(TME)以及免疫浸润的相关性。通过34个SERPIN基因确定了两个亚型,基于2个亚型筛选出了436个预后相关分型差异基因,通过Lasso回归确定出了11个预后相关基因参与风险模型的构建,根据模型评分区分的高低风险组具有明显的生存差异,列线图可以准确预测1、3和5年生存率。肿瘤微环境分析和免疫浸润分析显示高风险评分组患者免疫活性差。SERPIN家族相关基因构建的风险评分模型能够预测COAD的预后,有利于进一步指导临床对COAD的诊治,提高患者生存率。     

Overexpression of RBBP6, Alone or Combined with Mutant TP53, Is Predictive of Poor Prognosis in Colon Cancer

Retinoblastoma binding protein 6 (RBBP6) plays an important role in chaperone-mediated ubiquitination and interacts with TP53 in carcinogenesis. However, the clinicopathologic significance of RBBP6 expression in colon cancer is unknown; in particular, the prognostic value of RBBP6 combined with TP53 expression has not been explored. Therefore, quantitative real-time PCR and western blot analyses were performed to detect RBBP6 expression in colon cancer tissues. RBBP6 and TP53 expression were assessed by immunohistochemistry in a tissue microarray format, in which the primary colon cancer tissue was paired with noncancerous tissue. Tissue specimens were obtained from 203 patients. We found that RBBP6 was overexpressed in colon tumorous tissues and was significantly associated with clinical stage, depth of tumor invasion, lymph node metastasis (LNM), distant metastasis, and histologic grade. Further studies revealed that a corresponding correlation between RBBP6 overexpression and mutant TP53 was evident in colon cancer (r = 0.450; P<0.001). RBBP6 expression was an independent prognostic factor for overall survival (OS) and disease free survival (DFS). Interestingly, patients with tumors that had both RBBP6 overexpression and mutant TP53 protein accumulation relapsed and died within a significantly short period after surgery (P<0.001). Multivariate analysis showed that patients with LNM and patients with both RBBP6- and TP53-positive tumors had extremely poor OS (HR 6.75; 95% CI 2.63–17.35; P<0.001) and DFS (HR 8.08; 95% CI 2.80–23.30; P<0.001). These clinical findings indicate that the assessment of both RBBP6 and mutant TP53 expression will be helpful in predicting colon cancer prognosis.     

Enhanced expression of GABRD predicts poor prognosis in patients with colon adenocarcinoma

Neurotransmitters are reported to be involved in tumor initiation and progression. This study aimed to elucidate the prognostic value of γ-aminobutyric acid type A receptor δ subunit (GABRD) in colon adenocarcinoma (COAD) using the data from The Cancer Genome Atlas (TCGA) database. The GABRD mRNA expression levels in the COAD and normal tissues were compared using the Wilcoxon rank-sum test. The correlation between clinicopathologic characteristics and GABRD expression was analyzed by Wilcoxon rank-sum test or Kruskal-Wallis test and logistic regression. The prognostic value of GABRD mRNA expression in patients with COAD was determined using the Kaplan-Meier curve and Cox regression analysis. Finally, the molecular mechanisms of GABRD in COAD were predicted by gene set enrichment analysis (GSEA). The COAD tissues exhibited higher GABRD mRNA expression levels than the normal tissues. The logistic regression analysis revealed that GABRD mRNA expression was correlated with TNM stage, N stage, M stage, and microsatellite instability (MSI) status. The Kaplan-Meier survival curve and log-rank test revealed that patients with COAD exhibiting high GABRD mRNA expression were associated with poor overall survival (OS). The multivariate analysis indicated that increased GABRD mRNA expression was an independent prognostic factor and was correlated with a poor OS. The GSEA revealed that GABRD was involved in signaling pathways, including cell adhesion molecules, gap junction, melanogenesis, and mTOR signaling pathway, as well as the signaling pathways associated with basal cell carcinoma or bladder cancer development. In summary, enhanced GABRD mRNA expression may be a potential independent prognostic biomarker for COAD.     

7-Hydroxystaurosporine (UCN-01) preferentially sensitizes cells with a disrupted TP53 to gamma radiation in lung cancer cell lines

Mutations in TP53 occur in more than 50% of the lung cancer patients and are associated with an increased resistance to chemotherapy and radiotherapy. The human lung adenocarcinoma cell lines A549 and LXSN contain a wild-type TP53 and were growth arrested at both the G(1)- and G(2)-phase checkpoints after irradiation. However, a TP53-disrupted cell line, E6, was arrested only at the G(2)-phase checkpoint. UCN-01 (7-hydroxystaurosporine), a CHEK1 inhibitor that abrogates the G(2) block, has been reported to enhance radiation toxicity in human lymphoma and colon cancer cell lines. In this study, UCN-01 preferentially enhanced the radiosensitivity of the TP53-disrupted E6 cells compared to the TP53 wild-type cells. This effect was more pronounced in cells synchronized in early G(1) phase, where the E6 cells showed a higher resistance to radiation in the absence of drug. These results indicate that the combination of UCN-01 and radiation can more specifically target resistant TP53 mutated cancer cells and spare TP53 wild-type normal cells.     

Transcriptomic analysis of the effect of ifosfamide on MDCK cells cultivated in microfluidic biochips

We investigated the behavior of renal cells cultivated in microfluidic biochips when exposed to 50 μM of ifosfamide, an antineoplastic drug treatment. The microarray analysis revealed that ifosfamide had any effect in Petri conditions. The microfluidic biochips induced an early inflammatory response in the MDCK in the untreated cells. This was attributed to cells adapting to the dynamics and micro environment created by the biochips. This led to modulations in the mitochondria dysfunction pathway, the Nrf-2 and oxidative stress pathways and some related cancer genes. When exposed to 50 μM of ifosfamide, we detected a modulation of the pathways related to the cancer and inflammation in the MDCK cultivated in the biochips via modulation of the ATM, p53, MAP Kinase, Nrf-2 and NFKB signaling. In addition, the genes identified and related proteins affected by the ifosfamide treatment in the biochips such as TXNRD1, HSP40 (DNAJB4 and DNAJB9), HSP70 (HSPA9), p21 (CDKN1A), TP53, IKBalpha (NFKBIA) are reported to be the molecular targets in cancer therapy. We also found that the integrin pathway was perturbed with the ifosfamide treatment. Finally, the MYC proto-oncogene appeared to be a potential bridge between the integrin signaling and the anti-inflammatory response.     

Transcriptional landscape of DNA repair genes underpins a pan-cancer prognostic signature associated with cell cycle dysregulation and tumor hypoxia

Overactive DNA repair contributes to therapeutic resistance in cancer. However, pan-cancer comparative studies investigating the contribution of all DNA repair genes in cancer progression employing an integrated approach have remained limited. We performed a multi-cohort retrospective analysis to determine the prognostic significance of 138 DNA repair genes in 16 cancer types (n = 16,225). Cox proportional hazards analyses revealed a significant variation in the number of prognostic genes between cancers; 81 genes were prognostic in clear cell renal cell carcinoma while only two genes were prognostic in glioblastoma. We reasoned that genes that were commonly prognostic in highly correlated cancers revealed by Spearman’s correlation analysis could be harnessed as a molecular signature for risk assessment. A 10-gene signature, uniting prognostic genes that were common in highly correlated cancers, was significantly associated with overall survival in patients with clear cell renal cell (P < 0.0001), papillary renal cell (P = 0.0007), liver (P = 0.002), lung (P = 0.028), pancreas (P = 0.00013) or endometrial (P = 0.00063) cancers. Receiver operating characteristic analyses revealed that a combined model of the 10-gene signature and tumor staging outperformed either classifier when considered alone. Multivariate Cox regression models incorporating additional clinicopathological features showed that the signature was an independent predictor of overall survival. Tumor hypoxia is associated with adverse outcomes. Consistent across all six cancers, patients with high 10-gene and high hypoxia scores had significantly higher mortality rates compared to those with low 10-gene and low hypoxia scores. Functional enrichment analyses revealed that high mortality rates in patients with high 10-gene scores were attributable to an overproliferation phenotype. Death risk in these patients was further exacerbated by concurrent mutations of a cell cycle checkpoint protein, TP53. The 10-gene signature identified tumors with heightened DNA repair ability. This information has the potential to radically change prognosis through the use of adjuvant DNA repair inhibitors with chemotherapeutic drugs.     

DNAJB1 stabilizes MDM2 and contributes to cancer cell proliferation in a p53-dependent manner

Both MDM2 and MDMX regulate p53, but these proteins play different roles in this process. To clarify the difference, we performed a yeast 2 hybrid (Y2H) screen using the MDM2 acidic domain as bait. DNAJB1 was found to specifically bind to MDM2, but not MDMX, in vitro and in vivo. Further investigation revealed that DNAJB1 stabilizes MDM2 at the post-translational level. The C-terminus of DNAJB1 is essential for its interaction with MDM2 and for MDM2 accumulation. MDM2 was degraded faster by a ubiquitin-mediated pathway when DNAJB1 was depleted. DNAJB1 inhibited the MDM2-mediated ubiquitination and degradation of p53 and contributed to p53 activation in cancer cells. Depletion of DNAJB1 in cancer cells inhibited activity of the p53 pathway, enhanced the activity of the Rb/E2F pathway, and promoted cancer cell growth in vitro and in vivo. This function was p53 dependent, and either human papillomavirus (HPV) E6 protein or siRNA against p53 was able to block the contribution caused by DNAJB1 depletion. In this study, we discovered a new MDM2 interacting protein, DNAJB1, and provided evidence to support its p53-dependent tumor suppressor function.     

Clinical significance of preoperative serum sialic acid levels in colorectal cancer: utility in the detection of patients at high risk of tumor recurrence

This study was conducted to evaluate the significance of preoperative serum sialic acid levels in the diagnosis and prognosis of colorectal cancer (CRC). Total sialic acid (TSA) was determined by the thiobarbituric acid method and normalized to total protein (TP). A postoperative follow-up of CRC patients classified as Dukes' stages A, B or C was performed and survival analysis was carried out to evaluate the impact of sialic acid levels on tumor recurrence. Our diagnostic studies indicate that TSA/TP is a better marker than either TSA or carcinoembryonic antigen (CEA), especially for the detection of CRC patients at an early stage. At a cutoff of 30.90 nmol/mg of protein, TSA/TP showed a sensitivity of 85% with a specificity of 97% to discriminate CRC patients from healthy donors. In survival analysis, both TSA and TSA/TP were found to be significant prognostic factors for tumor recurrence in CRC. Furthermore, TSA/TP could distinguish patients at high risk of recurrence within Dukes' stage B and in multivariate analysis it was identified as the best independent prognostic factor. According to our results, preoperative serum TSA/TP content could supply additional information to that provided by Dukes' stage about the prognosis of CRC patients.     

Immune cell-derived small extracellular vesicles in cancer treatment

Small extracellular vesicles (sEVs) secreted by most cells carry bioactive macromolecules including proteins, lipids, and nucleic acids for intercellular communication. Given that some immune cell-derived sEVs exhibit anti-cancer properties, these sEVs have received scientific attention for the development of novel anti-cancer immunotherapeutic agents. In this paper, we reviewed the latest advances concerning the biological roles of immune cell-derived sEVs for cancer therapy. sEVs derived from immune cells including dendritic cells (DCs), T cells, natural-killer (NK) cells, and macrophages are good candidates for sEV-based cancer therapy. Besides their role of cancer vaccines, DC-shed sEVs activated cytotoxic lymphocytes and killed tumor cells. sEVs isolated from NK cells and chimeric antigen receptor (CAR) T cells exhibited cytotoxicity against cancer cells. sEVs derived from CD8⁺ T and CD4⁺ T cells inhibited cancer-associated cells in tumor microenvironment (TME) and activated B cells, respectively. M1-macrophage-derived sEVs induced M2 to M1 repolarization and also created a pro-inflammatory environment. Hence, these sEVs, via mono or combination therapy, could be considered in the treatment of cancer patients in the future. In addition, sEVs derived from cytokine-stimulated immune cells or sEV engineering could improve their anti-tumor potency.     

A correlation between BCL-2 modifying factor,p53 and livin gene expressions in cancer colon patients

Accumulating evidence has revealed that livin gene and BCL-2 modifying factor (BMF) gene are closely associated with the initiation and progression of colon carcinoma by activating or suppressing multiple malignant processes. Those genes that can detect colon - cancer are a promising approach for cancer screening and diagnosis. This study aimed to evaluate correlation between livin, BMF and p53 genes expression in colon cancer tissues of patients included in the study, and their relationship with clinicopathological features and survival outcome in those patients. In this study, 50 pathologically diagnosed early cancer colon patients included and their tissue biopsy with 50 matched adjacent normal tissue, and 50 adenoma tissue specimens were analyzed for livin gene and BMF gene expressions using real time PCR. The relationship of those genes expressions with clinicopathological features, tumor markers, Time to Progression and overall survival for those patients were correlated in cancer colon group. In this study, there was a significant a reciprocal relationship between over expression of livin gene and down regulation of BMF and p53 genes in colon cancer cells. Livin mRNA was significantly higher, while BMF and p53 mRNA were significantly lower in colorectal cancer tissue compared to benign and normal colon tissue specimens (P < 0.001), however, this finding was absent between colon adenomas and normal mucosa. There was a significant association between up regulation of livin and down regulation of BMF and p53 expressions with more aggressive tumor (advanced TNM stage), rapid progression with metastasis and decreased overall survival in cancer colon patients, hence these genes can serve as significant prognostic markers of poor outcome in colon cancer patients. This work highlights the role of livin, BMF and p53 genes in colorectal tumorigenesis and the applicability of using those genes as a diagnostic and prognostic markers in patients with colon carcinoma and as a good target for cancer colon treatment in the future.     

The clinical impact of miRNA34a and P53 gene expression in colon cancer

ObjectiveTo study the potential role of miRNA34a gene expression and its relationship with P53 gene expression, fate, stage, metastasis and overall survival of colorectal cancer.Patients and methodsThis study was carried out 30 patients with colon adenocarcinoma, 30 patients with benign colon polyp and 30 apparently healthy persons served as controls. All participants were subjected to full history taking, general clinical examination. Complete blood count, liver and kidney function, determination of serum tumor markers were done. Estimation of microRNA 34a and P53 Gene expression by real-time PCR were done.ResultsThere was a significant negative relationship between serum tumor markers and micro RNA 34a gene expression in cancer patients. Also, there was a statistically significant positive relationship between miRNA34a gene expression and P53 gene expression in both patients groups. The diagnostic accuracy of miRNA34a gene expression was both sensitive and specific for colon cancer. MiRNA34a and P53 gene expression had statistically significant relation with tumor stage and presence of metastases.ConclusionIt can be concluded that the level of miRNA34a can be used to differentiate between colon cancers and begin adenomas. MiRNA34a can be used as a prognostic marker in colon cancer.     

Serum anti-p53 antibodies as a useful marker for prognosis of gastric carcinoma

Mutations in the TP53 gene are the most common genetic alterations in cancer. Accumulation of mutated protein may induce circulating anti-p53 antibodies (anti-p53Ab) in sera of cancer patients. The aim of our work was to evaluate the presence and prognostic value of anti-p53Ab in gastric cancer patients and to investigate whether their presence is related to p53 overexpression in tumor tissue. Anti-p53Ab were analyzed in sera from 111 patients with gastric carcinoma and from 64 healthy donors by ELISA. p53 expression was also quantified by ELISA in biopsies of 54 gastric cancers and 22 healthy gastric mucosas. Significant anti-p53Ab levels were found in 15.3% of patients, whereas none of the 64 donor sera were positive. High levels of p53 expression were detected only in tumor tissue, in 72.2% of cases. A significant correlation was observed between anti-p53Ab and high levels of mutated p53 in tissue (p<0.05). The survival time of serum-positive patients was significantly longer than that of patients with low/negative serum levels, with a survival rate of 41.2% and 14.9%, respectively, over 48 months (p<0.05). Thus, detection of serum anti-p53Ab in gastric cancer patients can be useful to identify a subset of patients with better prognosis.     

Heat shock protein 27 as a prognostic and predictive biomarker in pancreatic ductal adenocarcinoma

A role of heat shock protein 27 (HSP27) as a potential biomarker has been reported in various tumour entities, but comprehensive studies in pancreatic cancer are lacking. Applying tissue microarray (TMA) analysis, we correlated HSP27 protein expression status with clinicopathologic parameters in pancreatic ductal adenocarcinoma specimens from 86 patients. Complementary, we established HSP27 overexpression and RNA-interference models to assess the impact of HSP27 on chemo- and radiosensitivity directly in pancreatic cancer cells. In the TMA study, HSP27 expression was found in 49% of tumour samples. Applying univariate analyses, a significant correlation was found between HSP27 expression and survival. In the multivariate Cox-regression model, HSP27 expression emerged as an independent prognostic factor. HSP27 expression also correlated inversely with nuclear p53 accumulation, indicating either protein interactions between HSP27 and p53 or TP53 mutation-dependent HSP27-regulation in pancreatic cancer. In the sensitivity studies, HSP27 overexpression rendered HSP27 low-expressing PL5 pancreatic cancer cells more susceptible towards treatment with gemcitabine. Vice versa, HSP27 protein depletion in HSP27 high-expressing AsPC-1 cells caused increased gemcitabine resistance. Importantly, HSP27 expression was inducible in pancreatic cancer cell lines as well as primary cells. Taken together, our study suggests a role for HSP27 as a prognostic and predictive marker in pancreatic cancer. Assessment of HSP27 expression could thus facilitate the identification of specific patient subpopulations that might benefit from individualized treatment options. Additional studies need to clarify whether modulation of HSP27 expression could represent an attractive concept to support the incorporation of hyperthermia in clinical treatment protocols for pancreatic cancer.     

TP53 and Let-7a micro-RNA Regulate K-Ras Activity in HCT116 Colorectal Cancer Cells

Recent reports have indicated that KRAS and TP53 mutations predict response to therapy in colorectal cancer. However, little is known about the relationship between these two common genetic alterations. Micro-RNAs (miRNAs), a class of noncoding RNA implicated in cellular processes, have been increasingly linked to KRAS and TP53. We hypothesized that lethal-7a (let-7a) miRNA regulates KRAS through TP53. To investigate the relationship between KRAS, TP53, and let-7a, we used HCT116 KRAS^mut human colorectal cancer cells with four different genotypic modifications in TP53 (TP53^−/−, TP53^+/−, TP53^mut/+, and TP53^mut/−). Using these cells we observed that K-Ras activity was higher in cells with mutant or knocked out TP53 alleles, suggesting that wild-type TP53 may suppress K-Ras activity. Let-7a was present in HCT116 KRAS^mut cells, though there was no correlation between let-7a level and TP53 genotype status. To explore how let-7a may regulate K-Ras in the different TP53 genotype cells we used let-7a inhibitor and demonstrated increased K-Ras activity across all TP53, thus corroborating prior reports that let-7a regulates K-Ras. To assess potential clinical implications of this regulatory network, we examined the influence of TP53 genotype and let-7a inhibition on colon cancer cell survival following chemoradiation therapy (CRT). We observed that cells with complete loss of wild-type TP53 alleles (^−/− or ^−/mut) were resistant to CRT following treatment with 5-fluorouracil and radiation. Further increase in K-Ras activity with let-7a inhibition did not impact survival in these cells. In contrast, cells with single or double wild-type TP53 alleles were moderately responsive to CRT and exhibited resistance when let-7a was inhibited. In summary, our results show a complex regulatory system involving TP53, KRAS, and let-7a. Our results may provide clues to understand and target these interactions in colorectal cancer.