X-ray crystallography study and optimization of novel benzothiophene analogs as potent selective estrogen receptor covalent antagonists (SERCAs) with improved potency and safety profiles.

Endocrine therapy (ET) is a well-validated strategy for estrogen receptor α positive (ERα + ) breast cancer therapy. Despite the clinical success of current standard of care (SoC), endocrine-resistance inevitably emerges and remains a significant medical challenge. Herein, we describe the structural optimization and evaluation of a new series of selective estrogen receptor covalent antagonists (SERCAs) based on benzothiophene scaffold. Among them, compounds 15b and 39d were identified as two highly potent covalent antagonists, which exhibits superior antiproliferation activity than positive controls against MCF-7 cells and shows high selectivity over ERα negative (ERα-) cells. More importantly, their mode of covalent engagement at Cys530 residue was accurately illustrated by a cocrystal structure of 15b-bound ERαY537S (PDB ID: 7WNV) and intact mass spectrometry, respectively. Further in vivo studies demonstrated potent antitumor activity in MCF-7 xenograft mouse model and an improved safety profile. Collectively, these compounds could be promising candidates for future development of the next generation SERCAs for endocrine-resistant ERα + breast cancer.

Novel 6-amino-1,3,5-triazine derivatives as potent BTK inhibitors: structure-activity relationship (SAR) analysis and preliminary mechanism investigation.

Bruton's tyrosine kinase (BTK) is a promising drug target for the treatment of B-cell related malignancies. Irreversible inhibition of BTK by a covalent inhibitor has been proved to be a clinically effective therapy. However, most irreversible BTK inhibitors also inhibit other kinases including JAK3 and EGFR, leading to some adverse events. Herein, we reported the structure-based design and optimization of a series of irreversible BTK inhibitors bearing the 6-amino-1,3,5-triazine scaffold. Most of the synthesized compounds demonstrated considerable BTK inhibition and improved anti-proliferative activity against Raji and Ramos cells. Among them, compound C11 exhibited potent BTK inhibition (BTK IC50 = 17.0 nM) and a desirable selectivity profile especially over EGFR. Moreover, C11 effectively blocked activation of BTK and downstream signaling, arrested the cell cycle in G0/G1 phase and induced apoptosis in Raji cells. Its irreversible binding mode was further investigated by both molecular modeling and a washout experiment. Collectively, C11 is a novel selective irreversible BTK inhibitor worthy of further in-depth research.

Development of novel tetrahydroisoquinoline-hydroxamate conjugates as potent dual SERDs/HDAC inhibitors for the treatment of breast cancer.

Concomitant inhibition of estrogen receptor alpha (ERα) and histone deacetylase (HDAC) signaling has been proven effective in endocrine-resistant ER+ breast cancers. Herein, a series of tetrahydroisoquinoline (THIQ)-hydroxamate conjugates were rationally designed and synthesized as dual SERDs/HDAC inhibitors by incorporating the hydroxamate, a known HDAC pharmacophore, into a privileged THIQ scaffold of selective ERα degraders (SERDs). Some of these THIQ-hydroxamate conjugates displayed remarkable HDAC6 inhibition and improved antiproliferative activity against MCF-7 cells. Particularly, the most potent HDAC inhibitor 19k also exhibits potent ERα binding affinity, good ERα degradation efficacy and the best antiproliferative activity. Besides, 19k displayed superior antitumor efficacy than the drug combination (Fulvestrant + SAHA) through promoting ERα degradation and histone acetylation in an MCF-7 xenograft model, without causing observable toxicity. Collectively, this study validates the therapeutic potential of a dual-acting compound with potent ERα degradation efficacy and HDAC6 inhibition in breast cancer.

Benzothiophene derivatives as selective estrogen receptor covalent antagonists: Design, synthesis and anti-ERα activities.

Estrogen receptor α emerged as a well validated therapeutic target of breast cancer for decades. However, approximately 50% of patients who initially responding to standard-of-care (SoC), such as undergo therapy of Tamoxifen, generally inevitably progress to an endocrine-resistance ER+ phenotype. Recently, selective estrogen receptor covalent antagonists (SERCAs) targeted to ERα have been demonstrated as a therapeutic alternative. In the present study, series of novel 6-OH-benzothiophene (BT) derivatives targeting ERα and deriving from Raloxifene were designed, synthesized, and biologically evaluated as covalent antagonists. Driven by the antiproliferative efficacy in ER+ breast cancer cells, our chemical optimization finally led to compound 19d that with potent antagonistic activity in ER+ tumor cells while without agonistic activity in endometrial cells. Moreover, the docking simulation was carried out to elucidate the binding mode, revealing 19d as an antagonist and covalently binding to the cysteine residue at the 530 position of ER helix H11.

Design and synthesis of novel benzothiophene analogs as selective estrogen receptor covalent antagonists against breast cancer.

Endocrine therapy (ET) has benefited patients with estrogen receptor alpha (ERα) positive breast cancer for decades. Selective estrogen receptor modulator (SERM) such as Tamoxifen represents the clinical standard of care (SoC). Despite the therapeutic importance of current SoC agents, 30-50% of prolonged treatment patients inevitably generated resistant tumor cells, usually eventually suffered tumor relapse and developed into metastatic breast cancer (MBC), which was the leading cause of female cancer-related mortality. Among these, most resistant tumors remained dependent on ERα signaling, which reignited the need for the next generation of ERα related agents. We hypothesized that selective estrogen receptor covalent antagonists targeting ERα would provide a therapeutic alternative. In the current work, series of novel benzothiophene hybrids bearing electrophile moieties were synthesized and biologically evaluated. The representative analogue 15c exhibited potent anti-proliferative effect in MCF-7 cell lines in vitro, and further mechanism studies confirmed the necessity of covalent bonding. More importantly, 15c could attenuate the expression of TFF-1, GREB-1 and downregulate the levels of cellular ERα protein.

Synthesis and evaluation of novel thiosemicarbazone and semicarbazone analogs with both anti-proliferative and anti-metastatic activities against triple negative breast cancer.

Triple-negative breast cancer (TNBC) is one of the most aggressive cancer with high mortality and recurrence rates. Hecogenin, a steroidal sapogenin, is reported as a potential anti-tumor agent against breast cancer. However, the moderate activity limits its further application in clinical. With the aim to identify novel analogues that are especially efficacious in therapy of TNBC, a series of novel hecogenin thiosemicarbazone and semicarbazone derivatives were designed, synthesized and biologically evaluated. Screening of cytotoxicity revealed that 4c could potently inhibit the proliferation of breast cancer cells (MCF-7 and MDA-MB-231 cells), lung cancer cells (A549) and colon cancer cells (HT-29) at low µM level. Importantly, further mechanism studies indicated the ability of 4c in inducing apoptosis of MDA-MB-231 cells by arresting the cell cycle. Moreover, 4c notably suppressed the migration and invasion of MDA-MB-231 cells compared to its parent hecogenin at the equal concentration.

Identification of Core Prognosis-Related Candidate Genes in Chinese Gastric Cancer Population Based on Integrated Bioinformatics.

BACKGROUND: Gastric cancer (GC) is one of the leading causes of cancer-related mortality worldwide. There are great geographical differences in the incidence of GC, and somatic mutation rates of driver genes are also different. The present study is aimed at screening core prognosis-related candidate genes in Chinese gastric cancer population based on integrated bioinformatics for the early diagnosis and prognosis of GC. METHODS: In the present study, the differentially expressed genes (DEGs) in GC were identified using four microarray datasets from the Gene Expression Omnibus (GEO) database. The samples of these datasets were all from China. Functional enrichment analysis of DEGs was conducted to evaluate the underlying molecular mechanisms involved in GC. Protein-protein interaction (PPI) network and cytoHubba were performed to determine hub genes associated with GC. Gene Expression Profiling Interactive Analysis (GEPIA) and Human Protein Atlas (HPA) were performed to validate the hub genes. RESULTS: A total of 240 DEGs were obtained through the RRA method, including 80 upregulated genes and 160 downregulated genes. Upregulated genes were mainly enriched in extracellular matrix organization, extracellular matrix, and extracellular matrix structural constituent. The downregulated genes were mainly enriched in digestion, extracellular space, and oxidoreductase activity. The KEGG pathway enrichment analysis showed that the upregulated genes were mainly associated with ECM-receptor interaction, focal adhesion, and PI3K-Akt signaling pathway. And downregulated genes were mainly associated with the metabolism of xenobiotics by cytochrome P450, metabolic pathways, and gastric acid secretion. The transcriptional and translational expression levels of the genes including COL1A1, COL5A2, COL12A1, and VCAN were higher in GC tissues than normal tissues. CONCLUSION: A total of four genes including COL1A1, COL5A2, COL12A1, and VCAN were considered potential GC biomarkers in the Chinese population. And ECM-receptor interaction, focal adhesion, and PI3K-Akt signaling pathway were revealed to be important mechanisms of GC. Our findings provide novel insights into the occurrence and progression of GC in the Chinese population.

Aberrant Expression of the Long Non-coding RNA GHRLOS and Its Prognostic Significance in Patients with Colorectal Cancer.

Long non-coding RNAs (lncRNAs), which have emerged as important regulatory RNA molecules that have been implicated in carcinogenesis and cancer progression, may also serve as novel potential biomarkers for cancer diagnosis and prognosis. Our previous analysis has identified the lncRNA GHRLOS, the ghrelin antisense strand non-coding RNA gene, as one of the hub genes in the co-expression network of differentially expressed lncRNAs/mRNAs in colorectal cancer (CRC). Here, we further evaluate the expression of GHRLOS in CRC and explore its clinical significance. The expression of GHRLOS in 366 pairs of CRC and adjacent non-cancerous tissues was detected by quantitative RT-PCR assays. The results showed that the expression level of GHRLOS was significantly lower in CRC tissues than in matched non-cancerous tissues (P < 0.001). Decreased GHRLOS expression was observed in 54.4% (199/366) of cases, and was significantly correlated with the occurrence of lymph node metastasis (P = 0.033) and distant metastasis (P = 0.005). A Kaplan-Meier analysis demonstrated that decreased GHRLOS expression contributed to poor disease-free survival (log-rank test, P < 0.001) and overall survival (log-rank test, P < 0.001). Moreover, a multivariate Cox regression analysis revealed the decreased expression of GHRLOS as an independent prognostic marker of poor outcomes [disease-free survival: hazard ratio (HR) = 2.02, 95% confidence interval (CI) = 1.42-3.88; overall survival: HR = 1.96, 95% CI = 1.34-2.86] in CRC patients. In conclusion, our data suggest that the lncRNA GHRLOS might serve as a candidate biomarker of tumor metastasis and a prognostic indicator in CRC.

Meta-analysis of SIRT1 expression as a prognostic marker for overall survival in gastrointestinal cancer.

Sirtuin 1 (SIRT1), a well-characterized NAD+-dependent histone deacetylase, is generally up-regulated in gastrointestinal cancers. However, the prognostic value of SIRT1 in gastrointestinal cancer remains inconclusive. Therefore, we report a meta-analysis of the association of SIRT1 expression with overall survival (OS) in gastrointestinal cancer. PubMed was systematically searched for studies evaluating the expression of SIRT1 and OS in patients with gastrointestinal cancer. Fifteen studies (six evaluating colorectal cancer, three evaluating hepatocellular carcinoma, three evaluating gastric cancer, and one each evaluating pancreatic cancer, esophageal squamous cell carcinoma, and gastroesophageal junction cancer) with 3,024 patients were finally included. The median percentage of gastrointestinal cancers with high SIRT1 expression was 52.5%. Overall analysis showed an association between high SIRT1 expression and worse OS [summary hazard ratio (sHR) 1.54, 95% confidence intervals (CI) 1.21-1.96] in gastrointestinal cancer. However, heterogeneity was observed across studies, which was mainly attributed to cancer type. Subgroup analysis revealed that SIRT1 was significantly associated with worse OS in non-colorectal gastrointestinal cancer (sHR 1.82, 95% CI 1.50-2.21), in particular in gastric cancer (sHR 3.19, 95% CI 1.97-5.16) and hepatocellular carcinoma (sHR 1.53, 95% CI 1.16-2.01), with no evidence of heterogeneity or bias. However, no association was observed in colorectal cancer (sHR 1.15, 95% CI 0.81-1.62). In conclusion, high SIRT1 expression is a potential marker for poor survival in non-colorectal gastrointestinal cancer, but not in colorectal cancer.

Association of obesity and overweight with overall survival in colorectal cancer patients: a meta-analysis of 29 studies.

PURPOSE: Previous studies that assessed the relationship between obesity, overweight, and survival in colorectal cancer (CRC) have provided conflicting results. Therefore, we quantitatively summarized existing evidence to estimate the association between obesity/overweight and overall survival (OS) in CRC patients and explored potentially important sources of variability. METHODS: Eligible studies were identified via PubMed and EMBASE searches. The summary hazard ratio (sHR) was estimated using a fixed-effects or random-effects model according to the heterogeneity between the studies. Meta-regression and subgroup analyses were performed to explore potential sources of heterogeneity. RESULTS: A total of 29 eligible studies, with 51,303 CRC patients, were finally included. The overall analysis showed worse OS among obese patients [sHR 1.10, 95 % confidence intervals (CI) 1.06-1.15], but not among overweight patients (sHR 0.92, 95 % CI 0.86-1.00), than in normal-weight patients. Considerable heterogeneity was observed across studies, which was primarily attributed to the timing of body mass index (BMI) assessment (meta-regression p < 0.05). The association between obesity and worse OS was strengthened when BMI was assessed before diagnosis (sHR 1.30, 95 % CI, 1.17-1.44). Conversely, post-diagnostic, in particular post-treatment, overweight was associated with a better OS (sHR 0.79, 95 % CI 0.70-0.91). Other factors, including gender, geographic location, and stage, may also modify the prognostic value of obesity or overweight. CONCLUSIONS: Obese but not overweight patients appear to have worse OS than normal-weight patients with CRC. The associations of obesity and overweight with OS in CRC patients majorly depend upon the timing of BMI assessment.

PIK3CA mutation is associated with poor survival among patients with metastatic colorectal cancer following anti-EGFR monoclonal antibody therapy: a meta-analysis.

PURPOSE: PIK3CA mutation appears to predict a lack of response to anti-EGFR monoclonal antibody (mAb) treatment in patients with metastatic colorectal cancer (mCRC). However, the predictive value of PIK3CA mutations for survival remains inconclusive. Here, we pooled the data from published studies to estimate the association between PIK3CA mutation and survival outcomes in mCRC patients treated with anti-EGFR mAbs. METHODS: Studies investigating the association of PIK3CA mutations with clinical survival outcomes of mCRC patients treated with anti-EGFR mAbs were systematically identified. The overall hazard ratio (HR) was estimated by using fixed effect model or random effect model according to heterogeneity between trails. RESULTS: Eight studies that reported survival outcome in 839 mCRC patients were included. In unselected patients, we found that PIK3CA mutations were significantly associated with poorer PFS [8 studies, 839 patients; HR = 1.53; 95 % confidence interval (CI) 1.28-1.84; P < 0.001] and OS (5 studies; 587 patients; HR = 1.28; 95 % CI 1.05-1.56; P = 0.015). With increased predictive power in KRAS wild-type patients (3 studies; 275 patients), the overall HR for PFS was 2.44 (95 % CI 1.33-4.48; P = 0.004) and was statistically significant. We also observed a worse OS in KRAS wild-type patients with PIK3CA mutations (2 studies; 163 patients; HR = 1.37; 95 % CI 0.80-2.35; P = 0.258), although the result was not statistically significant due to small sample size. CONCLUSIONS: PIK3CA mutation is a promising predictive biomarker for poor survival in mCRC patients treated with anti-EGFR mAbs, particularly in KRAS wild-type patients.

Preliminary screening and identification of stem cell-like sphere clones in a gallbladder cancer cell line GBC-SD.

This paper aims to screen and identify sphere clone cells with characteristics similar to cancer stem cells in human gallbladder cancer cell line GBC-SD. GBC-SD cells were cultured in a serum-free culture medium with different concentrations of the chemotherapeutic drug cisplatin for generating sphere clones. The mRNA expressions of stem cell-related genes CD133, OCT-4, Nanog, and drug resistance genes ABCG2 and MDR-1 in sphere clones were detected by quantitative real-time polymerase chain reaction (PCR). Stem cell markers were also analyzed by flow cytometry and immunofluorescent staining. Different amounts of sphere clones were injected into nude mice to test their abilities to form tumors. Sphere clones were formed in serum-free culture medium containing cisplatin (30 µmol/L). Flow cytometry results demonstrated that the sphere clones expressed high levels of stem cell markers CD133(+) (97.6%) and CD44(+) (77.9%) and low levels of CD24(+) (2.3%). These clones also overexpressed the drug resistance genes ABCG2 and MDR-1. Quantitative real-time PCR showed that sphere clones expressed stem cell genes Nanog and OCT-4 284 and 266 times, respectively, more than those in the original GBC-SD cells. Immunofluorescent staining showed that sphere clones overexpressed OCT-4, Nanog, and SOX-2, and low expressed MUC1 and vimentin. Tumor formation experiments showed that 1×10(3) sphere clone cells could induce much larger tumors in nude mice than 1×10(5) GBC-SD cells. In conclusion, sphere clones of gallbladder cancer with stem cell-like characteristics can be obtained using suspension cultures of GBC-SD cells in serum-free culture medium containing cisplatin.