Safety and efficacy of apatinib in patients with advanced gastric or gastroesophageal junction adenocarcinoma after the failure of two or more lines of chemotherapy (AHEAD): a prospective, single-arm, multicenter, phase IV study.

BACKGROUND: Apatinib, a highly selective VEGFR2 inhibitor, significantly improved efficacy versus placebo as a third- and later-line treatment for advanced gastric cancer in phase 2 and 3 trials. This prospective, single-arm, multicenter phase IV AHEAD study was conducted to verify the safety and efficacy of apatinib in patients with advanced or metastatic gastric or gastroesophageal adenocarcinoma after at least two lines of systematic therapy in clinical practice settings. METHODS: Patients with advanced gastric cancer who had previously failed at least two lines of chemotherapy received oral apatinib until disease progression, death or unacceptable toxicity. The primary endpoint was safety. The secondary endpoints included objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS) and overall survival (OS). Adverse events were summarized by the incidence rate. Median OS and PFS were estimated using the Kaplan-Meier method. ORR, DCR, OS at 3 and 6 months, and PFS at 3 and 6 months were calculated, and their 95% CIs were estimated according to the Clopper-Pearson method. RESULTS: Between May 2015 and November 2019, a total of 2004 patients were enrolled, and 1999 patients who received at least one dose of apatinib were assessed for safety. In the safety population, 87.9% of patients experienced treatment-related adverse events (TRAEs), with the most common hypertension (45.2%), proteinuria (26.5%), and white blood cell count decreased (25.3%). Additionally, 51% of patients experienced grade ≥ 3 TRAEs. Fatal TRAEs occurred in 57 (2.9%) patients. No new safety concerns were reported. Among the 2004 patients included in the intention-to-treat population, the ORR was 4.4% (95% CI, 3.6-5.4%), and DCR was 35.8% (95% CI, 33.7-38.0%). The median PFS was 2.7 months (95% CI 2.2-2.8), and the median OS was 5.8 months (95% CI 5.4-6.1). CONCLUSIONS: The findings in the AHEAD study confirmed the acceptable and manageable safety profile and clinical benefit of apatinib in patients with advanced gastric cancer as a third- or later-line of treatment. TRIAL REGISTRATION: This study was registered with ClinicalTrials.gov NCT02426034. Registration date was April 24, 2015.

DNA methylation-mediated repression of exosomal miR-652-5p expression promotes oesophageal squamous cell carcinoma aggressiveness by targeting PARG and VEGF pathways.

Exosomal microRNAs (miRNAs) have been recently shown to play vital regulatory and communication roles in cancers. In this study, we showed that the expression levels of miR-652-5p in tumour tissues and serum samples of oesophageal squamous cell carcinoma (OSCC) patients were lower compared to non-tumorous tissues and serum samples from healthy subjects, respectively. Decreased expression of miR-652-5p was correlated with TNM stages, lymph node metastasis, and short overall survival (OS). More frequent CpG sites hypermethylation in the upstream of miR-652-5p was found in OSCC tissues compared to adjacent normal tissues. Subsequently, miR-652-5p downregulation promoted the proliferation and metastasis of OSCC, and regulated cell cycle both in cells and in vivo. The dual-luciferase reporter assay confirmed that poly (ADP-ribose) glycohydrolase (PARG) and vascular endothelial growth factor A (VEGFA) were the direct targets of miR-652-5p. Moreover, the delivery of miR-652-5p agomir suppressed tumour growth and metastasis, and inhibited the protein expressions of PARG and VEGFA in nude mice. Taken together, our findings provide novel insight into the molecular mechanism underlying OSCC pathogenesis.

Correction: Molecular predictors of brain metastasis-related microRNAs in lung adenocarcinoma.

[This corrects the article DOI: 10.1371/journal.pgen.1007888.].

Molecular predictors of brain metastasis-related microRNAs in lung adenocarcinoma.

Brain metastasis (BM) is a major complication of lung adenocarcinoma (LAD). An investigation of the pathogenic mechanisms of BM, as well as the identification of appropriate molecular markers, is necessary. The aim of this study was to determine the expression patterns of microRNAs (miRNAs) in LAD with BM, and to investigate the biological role of these miRNAs during tumorigenesis. miRNA array profiles were used to identify BM-associated miRNAs. These miRNAs were independently validated in 155 LAD patients. Several in vivo and in vitro assays were performed to verify the effects of miRNAs on BM. We identified six miRNAs differentially expressed in patients with BM as compared to patients with BM. Of these, miR-4270 and miR-423-3p were further investigated. miR-4270 and miR-423-3p directly targeted MMP19 and P21, respectively, to influence cell viability, migration, and colony formation in vitro. miR-4270 downregulation and miR-423-3p upregulation was associated with an increased risk of BM in LAD patients. Thus, our results suggested that miR-4270 and miR-423-3p might play an important role in BM pathogenesis in LAD patients, and that these miRNAs might be useful prognostic and clinical treatment targets.

miR-3607-3p suppresses non-small cell lung cancer (NSCLC) by targeting TGFBR1 and CCNE2.

Accumulating evidence indicates that miRNAs can be promising diagnostic and/or prognostic markers for various cancers. In this study, we identified a novel miRNA, miR-3607-3p, and its targets in non-small cell lung cancer (NSCLC). The expression of miR-3607-3p was measured and its correlation with patient prognosis was determined. Ectopic expression in NSCLC cells, xenografts, and metastasis models was used to evaluate the effects of miR-3607-3p on proliferation and migration of NSCLC. Luciferase assay and western blotting were performed to validate the potential targets of miR-3607-3p after preliminary screening by microarray analysis and computer-aided algorithms. We demonstrated that miR-3607-3p was downregulated in NSCLC tissues and that miR-3607-3p might act as an independent predictor for overall survival in NSCLC. Moreover, serum miR-3607-3p may be a novel and stable marker for NSCLC. We found that overexpression of miR-3607-3p inhibited cell proliferation, colony formation, migration and invasion, and hampered the cell cycle of NSCLC cell lines in vitro. Our results suggested that miR-3607-3p directly targets TGFBR1 and CCNE2. In accordance with in vitro studies, we confirmed that miR-3607-3p functions as a potent suppressor miRNA of NSCLC. We showed that miR-3607-3p agomir could reduce tumor growth and inhibit TGFBR1 and CCNE2 protein expression. Taken together, our findings indicate that miR-3607-3p can inhibit NSCLC cell growth and metastasis by targeting TGFBR1 and CCNE2 protein expression, and provide new evidence of miR-3607-3p as a potential non-invasive biomarker and therapeutic target for NSCLC.

Epigenetically Upregulated MicroRNA-602 Is Involved in a Negative Feedback Loop with FOXK2 in Esophageal Squamous Cell Carcinoma.

MicroRNA is an endogenous, small RNA controlling multiple target genes and playing roles in various tumorigenesis processes. In this study, our results revealed that miR-602 expression levels in tumor tissues and preoperative serum from esophageal squamous cell carcinoma (ESCC) patients were higher than those in non-tumorous tissues and healthy volunteers. miR-602 overexpression was closely related to lymph node metastasis and TNM stages and correlated short overall, and it acted as an independent prognostic marker of ESCC. The methylation status of the miR-602 gene indicated more frequent hypomethylation of the CpG sites located upstream of the miR-602 gene in the ESCC tissues than in the adjacent normal tissues, and the methylation status of miR-602 correlated inversely with its expression levels. Subsequently, miR-602 overexpression promoted ESCC proliferation and metastasis and regulated cell cycles in vitro and in vivo. Mechanistically, a dual-luciferase experiment validated that Fork head box (FOX)K2 (FOXK2) was a direct target of miR-602. More importantly, systemic delivery of formulated miR-602 antagomir could reduce tumor growth and increased FOXK2 protein expression in nude mice. This work provides novel insight into the molecular pathogenesis of ESCC.

Proteasome-Independent Protein Knockdown by Small-Molecule Inhibitor for the Undruggable Lung Adenocarcinoma.

Therapeutic target identification and corresponding drug development is a demanding task for the treatment of lung adenocarcinoma, especially the most malignant proximal-proliferative subtype without druggable protein kinase mutations. Using a cell-SELEX-generated aptamer, we discovered a new tumor driver protein, leucine-rich pentatricopeptide repeat-containing protein (LRPPRC), which is specifically overexpressed in the most lethal subtype of lung adenocarcinoma. Targeted LRPPRC protein knockdown is a promising therapeutic strategy for the undruggable LUAD (lung adenocarcinoma). Nevertheless, LRPPRC is mainly located in mitochondria and degraded by protease. Current protein knockdown approaches, such as proteolysis-targeting chimeras (PROTACs), have limitations in their applications to the proteins degraded through proteasome-independent ways. Here, we designed an aptamer-assisted high-throughput method to screen small molecules that could bind to LRPPRC directly, disrupt the interaction of LRPPRC with its stabilizing chaperon protein, and lead to LRPPRC degradation by mitochondrial protease. The screened compound, gossypolacetic acid (GAA), is an old medicine that can accomplish the new function for targeted LRPPRC knockdown. It showed significant antitumor effects even with the LRPPRC-positive patient-derived tumor xenograft (PDX) model. This work not only extended the application of aptamers to screen small-molecule inhibitors for the undruggable lung cancers, but more importantly provided a new strategy to develop protein knockdown methods beyond the proteasome system.

P-Hydroxycinnamaldehyde Induces B16-F1 Melanoma Cell Differentiation via the RhoA-MAPK Signaling Pathway.

BACKGROUND/AIMS: Due to its antitumor and gastroprotective properties, cochinchina momordica seed (CMS), has been widely used to treat cancer patients in Asia. Our previous reports have shown that CMS is able to induce the differentiation of B16-F1 melanoma cells. However, its functional component and mechanism remain unclear and are addressed in this study. METHODS AND RESULTS: CMSP (p-hydroxycinnamaldehyde isolated from CMS) inhibited the proliferation, migration and invasiveness of B16-F1 cells both in vivo and in vitro. CMSP also induced the differentiation of B16-F1 cells, as characterized by dendrite-like outgrowth, increased melanogenesis and enhanced tyrosinase activity. Furthermore, CMSP treatment reduced the level of malignant markers of melanoma, specifically S-100B and melanoma-derived growth regulatory protein precursor (MIA), in a concentration-dependent manner. According to a western blot analysis, B16-F1 cells treated with CMSP exhibited a sustained increase in p-P38 and decreased activities of ERK and JNK. Our data further indicated that the downregulation of GTP-RhoA, which was mediated by increased cAMP release, was involved in CMSP-induced changes in MAPK, while LPA (Lysophosphatidic acid) partially reversed CMSP-induced B16 cell differentiation. CONCLUSION: These results demonstrated that CMSP-induced differentiation of B16F1 cells may occur through the RhoA-MAPK axis, which suggests a new potential strategy for melanoma treatment.

Proto-oncogene Wip1, a member of a new family of proliferative genes in NSCLC and its clinical significance.

This study aimed to analyze the expression, clinical significance of proto-oncogene in non small cell lung cancer (NSCLC), and the biological effect in its cell line by siRNA targeting wild-type p53-induced phosphatase 1 (Wip1). Immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) were, respectively, used to analyze Wip1 protein expression in 75 cases of NSCLC and normal tissues to study the relationship between Wip1 expression and clinical parameters. Wip1 siRNA was transiently transfected into papillary NSCLC H1299 cell by liposome-mediated method and was detected by RT-PCR and Western blot. MTT assay, cell apoptosis, and cell cycle were also conducted as to the influence of the downregulated expression of Wip1 that might be found on H1299 cells biological effect. The positive rates of Wip1 protein was 69.3% in NSCLC tissues but 16.0% expressed in normal tissues (P < 0.05). The relative content of Wip1 mRNA was 0.785 ± 0.062 and 0.147 ± 0.020 in NSCLC tissues and normal tissues, respectively, with significant differences between the two types (P < 0.05). There were no significant differences between Wip1 expression and sex, age, tumor size, and pathological types (P > 0.05). However, there were significant differences between Wip1 expression and lymph node metastasis, clinical stages, and tumor differentiation (P < 0.05). Individuals with positive and negative levels of Wip1 expression showed were statistically significant differences in the 5-year overall survival rate (P < 0.05). RT-PCR and Western blot showed that H1299 cell transfected Wip1 siRNA had a lower relative expressive content than normal cell (P < 0.05). MTT assay, cell apoptosis, and cell cycles demonstrated that H1299 cell transfected Wip1 siRNA had a lower survival fraction, higher cell apoptosis, more percentage of the G0/G1 phases, and lower cells in the S phases (P < 0.05). Wip1 protein and mRNA were increased in NSCLC, specifically in lymph node metastasis, clinical stages, and tumor differentiation. Wip1 may be involved in the biological processes of NSCLC cell proliferation, cell apoptosis, and cell cycle.

[Isoliquiritigenin Modulates the Effect of LINC01503 
on Lung Squamous Carcinoma Cells].

BACKGROUND: Isoliquiritigenin (ISL) is an important pharmacological constituent of Glycyrrhiza glabra, which possesses a range of physiological and pharmacological activities, as well as significant antitumor activity, and can be used as a potential drug for targeted cancer therapy. LINC01503 is an oncogene, which has been closely associated with the malignant biological processes of many cancers. The aim of this study was to investigate the effects of ISL on the proliferation, apoptosis, invasion and migration of lung squamous carcinoma cells by regulating LINC01503. METHODS: Plasma was collected from lung squamous carcinoma patients and healthy individuals treated at Tangshan People's Hospital from January 2021 to December 2022. The expression of LINC01503 in lung squamous carcinoma plasma, tissues and cells was detected by real-time quantitative fluorescence polymerase chain reaction (qRT-PCR). Lung squamous carcinoma cells were treated with different concentrations of ISL for 24 h, and LINC01503 expression was detected by qRT-PCR. The cells were treated in groups: si-NC group, si-LINC01503 group, DMSO (0.1% dimethyl sulfone) group, ISL group, pc DNA3.1(+)-NC group, pc DNA3.1(+)-LINC01503 group, ISL+pc DNA3.1(+)-NC group and ISL+pc DNA3.1(+)- LINC01503 groups. CCK-8 assay, clone formation assay, flow cytometry, Transwell assay and scratch assay were used to explore the effect of LINC01503 on the functional phenotype of lung squamous carcinoma cells. RESULTS: Fluorescence in situ hybridization results showed that the average fluorescence intensity of LINC01503 in tissue microarrays of lung squamous carcinoma patients was higher than that in paracancerous tissues (P<0.05). The expression of LINC01503 in the plasma of patients with lung squamous carcinoma was higher than that in the plasma of healthy individuals (P<0.05). Knockdown of LINC01503 inhibited the proliferation, invasion and migration of lung squamous carcinoma cells and promoted apoptosis (P<0.05). ISL inhibited the proliferation, invasion, migration and promoted apoptosis of lung squamous carcinoma cells (P<0.05). Overexpression of LINC01503 followed by intervention with ISL reversed the promotional effect of overexpression of LINC01503 on the proliferation, invasion and migration of lung squamous carcinoma cells as well as the inhibitory effect on apoptosis (P<0.05). CONCLUSIONS: LINC01503 was highly expressed in lung squamous carcinoma, and LINC01503 could promote the proliferation, invasion and migration of lung squamous carcinoma cells and inhibit the apoptosis, ISL could inhibit the proliferation, invasion and migration of lung squamous carcinoma cells and promote apoptosis of lung squamous carcinoma cells by regulating the expression of LINC01503.