Honokiol activates LKB1-miR-34a axis and antagonizes the oncogenic actions of leptin in breast cancer

Leptin, a major adipocytokine produced by adipocytes, is emerging as a key molecule linking obesity with breast cancer therefore, it is important to find effective strategies to antagonize oncogenic effects of leptin to disrupt obesity-cancer axis. Here, we examine the potential of honokiol (HNK), a bioactive polyphenol from Magnolia grandiflora, as a leptin-antagonist and systematically elucidate the underlying mechanisms. HNK inhibits leptin-induced epithelial-mesenchymal-transition (EMT), and mammosphere-formation along with a reduction in the expression of stemness factors, Oct4 and Nanog. Investigating the downstream mediator(s), that direct leptin-antagonist actions of HNK; we discovered functional interactions between HNK, LKB1 and miR-34a. HNK increases the expression and cytoplasmic-localization of LKB1 while HNK-induced SIRT1/3 accentuates the cytoplasmic-localization of LKB1. We found that HNK increases miR-34a in LKB1-dependent manner as LKB1-silencing impedes HNK-induced miR-34a which can be rescued by LKB1-overexpression. Finally, an integral role of miR-34a is discovered as miR-34a mimic potentiates HNK-mediated inhibition of EMT, Zeb1 expression and nuclear-localization, mammosphere-formation, and expression of stemness factors. Leptin-antagonist actions of HNK are further enhanced by miR-34a mimic whereas miR-34a inhibitor results in inhibiting HNK''s effect on leptin. These data provide evidence for the leptin-antagonist potential of HNK and reveal the involvement of LKB1 and miR-34a.     

The miR27b-CCNG1-P53-miR-508-5p axis regulates multidrug resistance of gastric cancer

Multidrug resistance (MDR) correlates with treatment failure and poor prognosis among gastric cancer (GC) patients. In a previous study using high-throughput functional screening, we identified 11 microRNAs (miRNAs) that regulate MDR in GC and found that miR-508-5p reversed MDR by targeting ABCB1 and ZNRD1. However, the mechanism by which miR-508-5p was decreased in chemo-resistant GC cells was unclear. In this study, we found that ectopic miR-27b is sufficient to sensitize tumors to chemotherapy in vitro and in vivo. Moreover, miR-27b directly targets the 3′ untranslated regions (3′-UTRs) of CCNG1, a well-known negative regulator of P53 stability. Interestingly, miR-27b up-regulation leads to increased miR-508-5p expression, and this phenomenon is mediated by CCNG1 and P53. Further investigation indicated that miR-508-5p is directly regulated by P53. Thus, the miR-27b/CCNG1/P53/miR-508-5p axis plays important roles in GC-associated MDR. In addition, miR-27b and miR-508-5p expression was detected in GC tissues with different chemo-sensitivities, and we found that tissues in which miR-27b and miR-508-5p are up-regulated are more sensitive to chemotherapy. Together, these data suggest that the combination of miR-27b and miR-508-5p represents a potential marker of MDR. Restoring the miR-27b and miR-508-5p levels might contribute to MDR reversion in future clinical practice.     

Nuclear osteopontin-c is a prognostic breast cancer marker

Background:

Although Osteopontin has been known as a marker for cancer progression, the elevated production of this cytokine is not specific for cancer. We have identified the splice variant Osteopontin-c as being absent from healthy tissue but associated with about 75% of breast cancer cases. However, in previous studies of Osteopontin-c, follow-up information was not available.

Methods:

Here we have analysed 671 patients, comprising a cohort of 291 paraffin blocks plus a population-based case-control study of 380 arrayed breast tumor tissues.

Results:

We find that high staining intensity of nuclear Osteopontin-c is strongly associated with mortality in patients with early breast cancer. Cytosolic staining for exon 4, reflective of Osteopontin-a and -b also predicts poor outcome. By contrast, total Osteopontin does not correlate with prognosis. These diverse assessments of Osteopontin also do not correlate with each other, suggesting distinct expression patterns for the variant forms. Consistent with its role in tumor progression, not tumor initiation, Osteopontin-c is not correlated with proliferation markers (Ki-67, cyclin A, cyclin B, cyclin E and cyclin D), neither is it correlated with ER, PR or HER2.

Conclusions:

The addition of Osteopontin-c immunohistochemistry to standard pathology work-ups may have prognostic benefit in early breast cancer diagnosis.     

Number of apoptotic cells as a prognostic marker in invasive breast cancer

Apoptosis plays an important role in tumorigenesis. Tumour growth is determined by the rate of cell proliferation and cell death. We counted the number of apoptotic cells in haematoxylin and eosin (H&E)-stained tumour sections in series of 172 grade I and II invasive breast cancers with long-term follow-up. The number of apoptotic cells in ten high-power fields were converted to the number of apoptotic cells per mm2 to obtain the apoptotic index (AI). The AI showed a positive correlation to the mitotic activity index (MAI) (P = 0.0001), histological grade (P < 0.0001) and worse tumour differentiation. Patients with high AI showed shorter overall survival than patients with low AI in the total group as well as in the lymph node-positive group. Tumour size, MAI, lymph node status and AI were independent prognostic indicators in multivariate analysis. The AI was shown to be of additional prognostic value to the MAI in the total patients group as well as in the lymph node-positive group. The correlation between the AI and the MAI points to linked mechanisms of apoptosis and proliferation. Since apoptotic cells can be counted with good reproducibility in H&E-stained tumour sections, the AI may be used as an additional prognostic indicator in invasive breast cancer.     

Quantitative assessment of miR34a as an independent prognostic marker in breast cancer

Background:

Aberrant expression of microRNAs (miRNAs) is associated with cancer progression, initiation and metastasis. MiR34a is a miRNA that has been previously described as a tumour suppressor. Herein, we assess the expression of miR34a in three independent breast cancer cohorts using a quantitative in situ hybridisation assay (qISH) and determined its association with disease-specific death in breast cancer.

Methods:

The qISH method was applied to three independent primary breast cancer cohorts (Cohort 1 with 461, Cohort 2 with 279 and Cohort 3 with 795 patients) using 5′ and 3′ double DIG-labelled LNA-modified probe against miR34a using the protocol described previously. Level of expression measured as automated quantitative analysis (AQUA) score for miR34a was determined for each patient and assessed for association with risk of disease-specific death. An optimal cutpoint was determined using the X-tile software for disease-specific survival in Cohort 1 and this cutpoint was then applied to the other two cohorts after median normalisation of AQUA scores.

Results:

Loss of miR34a is associated with poor outcome in three independent breast cancer cohorts (uncorrected log-rank P=0.0188 for Cohort 1, log-rank P=0.0024 for Cohort 2 and log-rank P=0.0455 for Cohort 3). In all three cohorts, loss of miR34a is able to stratify patients with poor disease-specific survival among node-negative patients, but not in node-positive population. Multivariate Cox proportional hazards analysis in Cohort 1 (P=0.0381) and Cohort 2 (P=0.0468) revealed that loss of miR34a is associated with poor outcome, independent of age, node status, receptor status and tumour size.

Conclusion:

Loss of the tumour suppressor, miR34a, identifies a subgroup of breast cancer patients with poor disease-specific survival. This study is consistent with the well-established preclinical observations for miR34a as a tumour suppressor and suggests that miR34a may have future value as a biomarker in breast cancer.     

The hsa_circ_0007396-miR-767-3p-CHD4 axis is involved in the progression and carcinogenesis of gastric cancer

BackgroundCircular RNAs (circRNAs) have been linked to numerous human cancers, including gastric cancer (GC), in numerous recent investigations. The expression of circRNA and the mechanisms involved in GC are still unknown.MethodsIn this work, Gene Expression Omnibus 2R (GEO2R) online tool was first used to screen 6 candidates of differentially expressed circRNAs in 2 datasets, {"type":"entrez-geo","attrs":{"text":"GSE83521","term_id":"83521"}}GSE83521 and {"type":"entrez-geo","attrs":{"text":"GSE89143","term_id":"89143"}}GSE89143. Then, using Cancer-Specific CircRNA Database (CSCD), the structural loop diagrams of these circRNAs were generated. After combining the Circular RNA Interactome (CRI) and CSCD databases for miRNA co-prediction, a candidate circRNA-miRNA sub-network was successfully created. The expression of these miRNAs was further examined using Cytoscape software, and 2 miRNAs, miR-767-5p and miR-767-3p.ResultsWe used GEO2R to analyze the differential expression of {"type":"entrez-geo","attrs":{"text":"GSE83521","term_id":"83521"}}GSE83521 and {"type":"entrez-geo","attrs":{"text":"GSE89143","term_id":"89143"}}GSE89143 datasets in GEO database. Through the construction of the structural ring diagram of CSCD database, we found that hsa_circRNA_100571, hsa_circRNA_103102, hsa_circRNA_100754, hsa_circRNA_100737, hsa_circRNA_100269, hsa_circRNA_102476, hsa_circRNA_101287 is the final candidate circRNA in GC. MiR-767-5p and miR-767-3p were found to be important miRNAs in GC. The miRNet database indicated their downstream target genes. In various studies, namely central gene screening, correlation analysis, and protein-protein interaction (PPI), we detected chromodomain helicase DNA binding protein 4 (CHD4) as a key potential candidate of hsa-mir-767-3p. Next, we conducted validation of clinical data. We included the clinical data of 100 patients with GC, and found that patients with low CHD4 expression had significantly higher OS and PFS than those with high CHD4 expression (P<0.001, P=0.005). Cox regression analysis showed that low CHD4 expression was an independent risk factor for tumor progression (P=0.001). At the same time, tumor differentiation and chemotherapy also had a certain impact on the progression of GC (all P<0.05). Therefore, CHD4 may provide a promising therapeutic target for the future treatment of GC.ConclusionsWe identified an important hsa_circ_0007396-miR-767-3p-CHD4 axis, which is associated with GC proliferation and carcinogenesis, and may represent a promising therapeutic target for the future cure of GC.     

TYMS-TM4SF4 axis promotes the progression of colorectal cancer by EMT and upregulating stem cell marker

Background: The expression of thymidylate synthase (TYMS) is significantly up-regulated in various cancers and associated with the poor prognosis of patients. However, the role of TYMS in the progression of colorectal cancer (CRC) is unclear. Methods: Cell function assay, biology information analysis, and RNA sequencing were used to investigate the role of TYMS in the progression of CRC and underlining molecular mechanism. SPSS22.0 statistical software and GraphPad Prism 5 (Graphpad software) were used for statistical analysis. Results: Our results showed that TYMS expression was higher in CRC tissues than that in non-tumor colorectal mucosa tissues. TYMS knockdown inhibited the proliferation, migration and invasion of HCT116 and HT29 cells, and the spheroid formation of HCT116 cells. The underling mechanism demonstrated that TYMS promoted the progression of CRC by regulating EMT-related proteins including E-cadherin, Vimentin, MMP-9 and stem cell biomarkers including CD133 and CD44. Furthermore, DEG sequencing showed that TYMS knockdown enriched the pathways of metastasis and metabolism by GO and KEGG analysis. We identified TM4SF4 was the downstream target of TYMS in CRC cells. TM4SF4 overexpression increased migration and invasion of CRC cells by regulating EMT and CD133 expression. Conclusions: Our findings suggest that TYMS-TM4SF4 axis may promote the progression of CRC by EMT and upregulating stem cell markers.     

Plasma hyaluronic acid level as a prognostic and monitoring marker of metastatic breast cancer

Conventional tumor markers have limited value for prognostication and treatment monitoring in metastatic breast cancer (MBC) patients and novel circulating tumor markers therefore need to be explored. Hyaluronic acid (HA) is a major macropolysaccharide in the extracellular matrix and is reported to be associated with tumor progression. In our study, we investigated plasma HA level with respect to progression free survival (PFS) and overall survival (OS), as well as the treatment monitoring value in MBC patients. The prognostic value of plasma HA level was investigated in a discovery cohort of 212 MBC patients with 2.5‐year follow‐up and validated in an independent validation cohort of 334 patients with 5‐year follow‐up. The treatment monitoring value of plasma HA level was investigated in 61 MBC patients from discovery cohort who had been radiographically examined after first complete cycle of chemo therapy. We found a robust association between high plasma HA level and poor prognosis of MBC patients in both discovery (p_PFS = 7.92 × 10^?6 and p_OS = 5.27 × 10^?5) and validation studies (p_PFS = 3.66 × 10^?4 and p_OS = 1.43 × 10^?4). In the discovery cohort, the plasma HA level displayed independent prognostic value after adjusted for age and clinicopathological factors, with respect to PFS and OS. Further, the decrease of plasma HA level displayed good concordance with treatment response evaluated by radiographic examination (AUC = 0.79). Plasma HA level displays prognostic value, as well as treatment monitoring value for MBC patients.     

miR-30e* is an independent subtype-specific prognostic marker in breast cancer

Background:

Breast cancer clinical outcome is affected by tumor molecular features, and the identification of subtype-specific prognostic biomarkers is relevant for breast cancer translational research. Gene expression signatures proved to be able to complement prognostic information provided by classical clinico-pathological features. Recently, microRNAs (miRNAs) have been causally linked to tumorigenesis and cancer progression and have been associated with patient outcome, also in breast cancer.

Methods:

MicroRNAs associated with the development of distant metastasis were identified in a cohort of 92 ESR1+/ERBB2− lymph node-negative breast cancers from patients not receiving adjuvant treatment. Results were confirmed and further investigated in a total of 1246 miRNA and gene expression profiles of the Molecular Taxonomy of Breast Cancer International Consortium data set. Moderated t-test, univariable and multivariable Cox regression models were used for statistical analyses.

Results:

miR-30e* was identified as independent protective prognostic factor in lymph node-negative untreated patients with ESR1+/ERBB2− tumours and retained a significant association with a good prognosis in treated patients with the same tumor subtype as well as in the ERBB2+ subtype, but not in ESR1−/ERBB2− tumours.

Conclusions:

We highlighted a relevant and subtype-specific role in breast cancer for miR-30e* and demonstrated that adding miRNA markers to gene signatures and clinico-pathological features can help for a better prognostication.     

Validation of UBE2C protein as a prognostic marker in node-positive breast cancer

Background:

We recently identified and validated UBE2C RNA as a prognostic marker in 252 node-positive (N+) breast cancers by means of a microarray study. The aim of this study was to validate UBE2C protein as a prognostic marker in N+ breast cancer by immunohistochemistry (IHC).

Methods:

To this end, 92 paraffin-embedded blocks were used. The impact of UBE2C IHC value on metastasis-free survival (MFS) and overall survival (OS) was evaluated and compared with Ki-67 and Nottingham prognostic index (NPI) performances.

Results:

In accordance with genomic data, UBE2C IHC had a significant impact both on MFS and OS (hazard ratio=6.79 – P=0.002; hazard ratio=7.14 – P=0.009, respectively). Akaike information criterion proved that the prognostic power of UBE2C IHC was stronger than that of Ki-67 (and close to that of NPI). Furthermore, multivariate analyses with NPI showed that, contrary to Ki-67 IHC, UBE2C IHC remained an independent factor, both for MFS (adjusted P=0.02) and OS (adjusted P=0.04).

Conclusion:

We confirmed that UBE2C protein measured by IHC could be used as a prognostic marker in N+ breast cancer. The potential predictive interest of UBE2C as a marker of proteasome activity needs further investigations.     

lncRNA XIST通过调控miR-337-3p/HOXC8 轴促进胃癌的发展进程

[摘要] 目的：探讨lncRNA XIST（XIST）通过调控miR-337-3p/HOXC8 分子轴介导胃癌发展进程的分子机制。方法：选取2013 年3 月至2018 年1 月河北省唐山市开滦总医院普通外科手术切除的58 例胃癌组织和对应的癌旁组织标本,以及人胃癌细胞系AGS、MGC803、HGC27 和人胃黏膜细胞GES-1,用qPCR检测胃癌组织和细胞系中XIST 和miR-337-3p 的表达水平。将XIST敲降载体、miR-337-3p mimics/inhibitor 和HOXC8 过表达载体转染进AGS细胞,用CCK-8、Transwell 实验检测AGS细胞的增殖及侵袭能力,用WB检测HOXC8 及上皮钙黏蛋白（E-cadherin）、神经钙黏蛋白（N-cadherin）和波形蛋白（vimentin）的表达水平。用双荧光素酶报告基因验证XIST、miR-337-3p 和HOXC8 的靶向关系。结果：XIST在胃癌组织和细胞系中高表达（均P<0.01）。敲降XIST 显著抑制AGS细胞的增殖、侵袭和EMT（P<0.05 或P<0.01）。XIST 靶向作用miR-337-3p 并下调其表达,HOXC8 是miR-337-3p 的靶基因。敲降XIST 通过靶向上调miR-337-3p 对HOXC8 表达的抑制作用,从而抑制AGS细胞增殖、侵袭和EMT（P<0.05 或P<0.01）。结论：敲降XIST 可抑制AGS 细胞增殖、侵袭和EMT,其作用机制是通过靶向miR-337-3p 并下调HOXC8的表达。     

Cell kinetics as a prognostic marker in node-negative breast cancer

The relevance of cell kinetics of the primary tumor as a marker of biologic aggressivity was verified on a series of 258 patients with operable breast cancer without nodal and distant metastases, who underwent radical mastectomy without any other treatment until relapse. The cell proliferative rate, evaluated in vitro on fresh tumor material and expressed as 3H-thymidine labeling index, was found to be an important discriminant of metastatic potential. Patients with slowly proliferating tumors showed a higher probability of 6-year relapse-free survival in comparison to those with fast-proliferating tumors (80.5% versus 59.6%, P = 0.00004). This finding was true for premenopausal and paramenopausal (P = 0.00005) as well as for postmenopausal patients (P = 0.01). The kinetic variable was also a discriminant of 6-year survival for the overall series (P = 0.003) and for premenopausal and paramenopausal patients (P = 0.003) regardless of the type of treatment after relapse. These findings show the relevance of the kinetic variable as an important prognostic marker in breast cancer.     

ING5 inhibits cancer aggressiveness via preventing EMT and is a potential prognostic biomarker for lung cancer

The proteins of the Inhibitor of Growth (ING) candidate tumor suppressor family are involved in multiple cellular functions such as cell cycle regulation, apoptosis, and chromatin remodeling. ING5 is the new member of the family whose actual role in tumor suppression is not known. Here we show that ING5 overexpression in lung cancer A549 cells inhibited cell proliferation and invasiveness, while ING5 knockdown in lung cancer H1299 cells promoted cell aggressiveness. ING5 overexpression also abrogated tumor growth and invasive abilities of lung cancer cells in mouse xenograft models. Further study showed that ING5 overexpression inhibited EMT indicated by increase of E-cadherin and decrease of N-cadherin, Snail and slug at mRNA and protein levels, which was accompanied with morphological changes. cDNA microarray and subsequent qRT-PCR validation revealed that ING5 significantly downregulated expression of EMT (epithelial to mesenchymal transition)-inducing genes including CEACAM6, BMP2 and CDH11. Clinical study by tissue microarray showed that nuclear ING5 negatively correlated with clinical stages and lymph node metastasis of lung cancer. Furthermore, high level of nuclear ING5 was associated with a better prognosis. Taken together, these findings uncover an important role for ING5 as a potent tumor suppressor in lung cancer growth and metastasis.     

RCAS1 as a tumour progression marker: an independent negative prognostic factor in gallbladder cancer.

Receptor-binding cancer antigen expressed on SiSo cells (RCAS1) induces apoptosis in immune cells bearing the RCAS1 receptor. We sought to determine RCAS1 involvement in the origin and progression of gallbladder cancer, and also implications of RCAS1 for patient survival. RCAS1 expression was examined immunohistochemically in 110 surgically resected gallbladder specimens. The gallbladders represented 20 cases of cholecystitis with no associated pancreaticobiliary maljunction; 23 cases of cholecystitis with pancreaticobiliary maljunction; 14 cases of adenomyomatosis; 7 adenomas; and 46 cancers. High expression of RCAS1 (immunoreactivity in over 25% of cells) was observed in 32 of the 46 cancers (70%), but not in other diseases, including pre-cancerous conditions. RCAS1 immunoreactivity was associated with depth of tumour invasion (P = 0.0180), lymph node metastasis (P = 0.0033), lymphatic involvement (P = 0.0104), venous involvement (P = 0.0224), perineural involvement (P = 0.0351) and stage by the tumour, nodes and metastases (TNM) classification (P = 0.0026). Thus, RCAS1 expression may be a relatively late event in gallbladder carcinogenesis, possibly promoting tumour progression. Cox regression multivariate analysis demonstrated RCAS1 positivity to be an independent negative predictor for survival (P = 0.0337; risk ratio, 12.690; 95% confidence interval, 1.216-132.423). High expression of RCAS1 significantly correlated with tumour progression and predicted poor outcome in gallbladder cancer.     

Aneuploidy as a prognostic factor in breast cancer

The predictive value of nuclear DNA content in mammary carcinoma is still under debate in spite of several reports indicating a relationship between DNA ploidy and prognosis. The impact of differences in methodology on the evaluation of DNA data is discussed, and a recent study demonstrating DNA ploidy as a statistically significant prognostic variable on a prospective material of breast cancer patients is presented.