To investigate the effect of RAS on anti-EGFR moAb + 5-FU infusion based chemotherapy in first-line treatment of mCRC.

The epithelial-to-mesenchymal transition (EMT) is a well-known prerequisite for cancer cells to acquire the migratory and invasive capacity, and to subsequently metastasize. Bufalin is one of the major active components of the traditional Chinese medicine Chan Su, and accumulating evidence has shown its anticancer effect in multipe types of cancer. However, the role of bufalin in transforming growth factor‑β (TGF‑β)‑induced EMT and migration remains unclear. In the present study, the effect of bufalin on TGF‑β‑induced EMT and migration was investigated in human lung cancer A549 cells. TGF‑β induced EMT in A549 cells and increased their migratory ability, which were markedly suppressed by bufalin. Additionally, TGF‑β‑induced upregulation of Twist2 and zinc finger E‑box binding homeobox 2 (ZEB2), as well as the phosphorylation of Smad2 and Smad3 were also inhibited by bufalin. However, the Smad‑independent signaling pathways were not affected. Further analysis showed that the TGF‑β receptor I (TβRI) and TGF‑β receptor II (TβRII) were downregulated in the presence of bufalin. Pretreatment with SB431542, a potent inhibitor of the phosphorylation of TβRI, significantly attenuated TGF‑β‑induced EMT, mimicking the effect of bufalin on A549 cells. Taken together, these results suggest that bufalin suppresses TGF-β-induced EMT and migration by downregulating TβRI and TβRII in A549 cells.

Although anti-programmed death ligand-1 (PD-L1) therapy has shown light in treatment of gastric cancer, only a limited number of patients respond to the treatment. In addition to its immunosuppressive effect, PD-L1 is involved in other functions of tumor cells. Previously study showed that PD-L1 promoted EMT in lung cancer cells. However, the other effect and role of PD-L1 in gastric cancer remains unclear. In the present study, we first demonstrated that PD-L1 promoted the proliferation and migration in gastric cancer cell lines. We found that another STAT family member, STAT5a, is involved in regulating the expression of PD-L1 in gastric cancer. Additionally, Cbl-b interacted and ubiquitated STAT5a, down-regulated the expression of STAT5a and PD-L1. Moreover, bioinformatics predictions and experimental data showed that Cbl-b is a target gene of the microRNA miR-940. We further found that miR-940 promoted the proliferation and migration of gastric cancer in vivo and in vitro. Taken together, our findings suggest that miR-940/Cbl-b/STAT5a axis regulated the expression of PD-L1, which promotes the proliferation and migration of gastric cancer cells.

Missense mutation is the most common mutation type in hemophilia. However, the majority of missense mutations remain uncharacterized. Here we characterize how hemophilia mutations near the unused N-glycosylation site of the A2 domain (N582) of FVIII affect protein conformation and intracellular trafficking. N582 is located in the middle of a short 310-helical turn (D580-S584), in which most amino acids have multiple hemophilia mutations. All 14 missense mutations found in this 310-helix reduced secretion levels of the A2 domain and full-length FVIII. Secreted mutants have decreased activities relative to WT FVIII. Selected mutations also lead to partial glycosylation of N582, suggesting that rapid folding of local conformation prevents glycosylation of this site in wild-type FVIII. Protease sensitivity, stability and degradation of the A2 domain vary among mutants, and between non-glycosylated and glycosylated species of the same mutant. Most of the mutants interact with the ER chaperone BiP, while only mutants with aberrant glycosylation interact with calreticulin. Our results show that the short 310-helix from D580 to S584 is critical for proper biogenesis of the A2 domain and FVIII, and reveal a range of molecular mechanisms by which FVIII missense mutations lead to moderate to severe hemophilia A.

The ATR/checkpoint kinase 1 (Chk1) pathway plays an essential role in modulating the DNA damage response and homologous recombination. Particularly, Chk1 phosphorylation is related to cancer prognosis and therapeutic resistance. Some receptor tyrosine kinases participate in the regulation of Chk1 phosphorylation; however, the effect of hepatocyte growth factor (HGF) on Chk1 phosphorylation is unknown. In the present study, we demonstrated that HGF moderately activated Chk1 phosphorylation in colon cancer cells by upregulating TopBP1 and RAD51, and promoting TopBP1-ATR complex formation. Furthermore, AKT activity, which was promoted by HGF, served as an important mediator linking HGF/MET signaling and Chk1 phosphorylation. Depleting AKT activity attenuated basal expression of p-Chk1 and HGF-induced Chk1 activation. Moreover, AKT activity directly regulated TopBP1 and RAD51 expression. AKT inhibition suppressed HGF-induced upregulation of TopBP1 and RAD51, and enhanced TopBP1/ATR complex formation. Our results show that HGF was involved in regulating Chk1 phosphorylation, and further demonstrate that AKT activity was responsible for this HGF-induced Chk1 phosphorylation. These findings might potentially result in management of prognosis and therapeutic sensitivity in cancer therapy.

The abnormal m6A modification caused by m6A modulators is a common feature of various tumors; however, little is known about which m6A modulator plays the most important role in triple-negative breast cancer (TNBC). In this study, when analyzing the influence of m6A modulators (METTL3, METTL14, WTAP, FTO, and ALKBH5) on the prognosis of breast cancer, especially in TNBC using several on-line databases, methyltransferase-like 3 (METTL3) was found to have low expression in breast cancer, and was closely associated with short-distance-metastasis-free survival in TNBC. Further investigation showed that knockdown of METTL3 could enhance the ability of migration, invasion, and adhesion by decreasing m6A level in TNBC cell lines. Collagen type III alpha 1 chain (COL3A1) was identified and verified as a target gene of METTL3. METTL3 could down-regulate the expression of COL3A1 by increasing its m6A methylation, ultimately inhibiting the metastasis of TNBC cells. Finally, with immunohistochemistry staining in breast cancer tissues, it was proved that METTL3 expression was negatively correlated with COL3A1 in TNBC, but not in non-TNBC. This study demonstrated the potential mechanism of m6A modification in metastasis and provided potential targets for treatment in TNBC.

Gastric cancer (GC) is a common cause of cancer-related death worldwide. As a result of the lack of reliable diagnostic or prognostic biomarkers for GC, patient prognosis is still poor. Therefore, there is an urgent need for studies examining the underlying pathogenesis of GC in order to find effective biomarkers. LRRN1 (leucine-rich repeat neuronal protein-1) is a type I transmembrane protein that plays an important role in the process of nerve development and regeneration. However, its role in cancer, especially in GC, remains unclear. In the present study, we found that LRRN1 expression is upregulated in GC tissues and that high LRRN1 expression is associated with poor prognosis. siRNA and shRNA-mediated knockdowns of LRRN1 expression promoted GC cell apoptosis and activation of the Fas/FasL pathway. LRRN1 knockdown also resulted in upregulation of JUN, a subunit of the transcription factor AP-1 (activator protein-1). This suggests that LRRN1 suppresses GC cell apoptosis by downregulating AP-1, resulting in inhibition of the Fas/FasL pathway. These results confirm that LRRN1 plays a significant role in GC pathogenesis. Moreover, LRRN1 may be a potential prognostic biomarker and therapeutic target for GC.

Non-small-cell lung cancer (NSCLC) is the leading global cause of cancer-related death. Due to the lack of reliable diagnostic or prognostic biomarkers, the prognosis of NSCLC remains poor. Consequently, there is an urgent need to explore the mechanisms underlying this condition in order to identify effective biomarkers. G-protein-signaling modulator 2 (GPSM2) is widely recognized as a determinant of mitotic spindle orientation. However, its role in cancer, especially NSCLC, remains uncertain. In this study, we found that GPSM2 was downregulated in NSCLC tissues and was correlated with a poor prognosis. Furthermore, the knockdown of GPSM2 promoted NSCLC cell metastasis in vitro and in vivo and accelerated the process of epithelial-mesenchymal transition (EMT). Mechanistically, we showed that silencing GPSM2 induced cell metastasis and EMT through the ERK/glycogen synthase kinase-3β/Snail pathway. These results confirm that GPSM2 plays an important role in NSCLC. Moreover, GPSM2, as an independent prognostic factor, could be a potential prognostic biomarker and drug target for NSCLC.

BACKGROUND Serine proteinase inhibitor clade B member 3 (SERPINB3) is a neutral glycoprotein. Its overexpression is related to the promotion of cell proliferation and activation via the nuclear factor kappa B (NF-kappaB) pathway in several tumors. Whether it can participate in stromal cell-derived factor (SDF-1)/NF-kappaB-induced metastasis of gastric cancer has not been reported. MATERIAL AND METHODS We analyzed the ability of SDF-1 to induce migration and invasion in vitro by knocking down the expression of SERPINB3 with siRNAs in gastric cancer cells. We also explored the effects of a CXCR4 antagonist and NF-kappaB inhibitor on SERPINB3 expression. We verified the effect of SERPINB3 on prognosis in gastric cancer specimens by immunohistochemistry. RESULTS In vitro experiments confirmed that SDF-1 upregulated the expression of SERPINB3 and promoted metastasis in gastric cancer cells. This phenomenon was reversed by knockdown of SERPINB3, a chemokine receptor 4 (CXCR4) antagonist, and an NF-kappaB inhibitor, which downregulated the expression of SERPINB3. In patients with gastric cancer, a significant positive correlation was observed between CXCR4 and SERPINB3 expression (r=0.222, P=0.029). Moreover, double positivity for SERPINB3 and CXCR4 was certified to be an independent prognostic factor (HR=3.332, P<0.001). CXCR4-positive patients who also expressed SERPINB3 were inclined to suffer from lymph node metastasis, confirming that SERPINB3 is a downstream molecule of CXCR4. CONCLUSIONS In vitro and pathological results showed that SDF-1/CXCR4 activated the NF-kappaB pathway and upregulated SERPINB3 to facilitate the migration and invasion of gastric cancer cells.

α1-antitrypsin (AAT) is a serine protease inhibitor synthesized in hepatocytes and protects the lung from damage by neutrophil elastase. AAT gene mutations result in AAT deficiency (AATD), which leads to lung and liver diseases. The AAT Z variant forms polymer within the endoplasmic reticulum (ER) of hepatocytes and results in reduction in AAT secretion and severe disease. Previous studies demonstrated a secretion defect of AAT in LMAN1 deficient cells, and mild decreases in AAT levels in male LMAN1 and MCFD2 deficient mice. LMAN1 is a transmembrane lectin that forms a complex with a small soluble protein MCFD2. The LMAN1-MCFD2 protein complex cycles between the ER and the Golgi. Here, we report that LMAN1 and MCFD2 knockout (KO) HepG2 and HEK293T cells display reduced AAT secretion and elevated intracellular AAT levels due to a delayed ER-to-Golgi transport of AAT. Secretion defects in KO cells were rescued by wild-type LMAN1 or MCFD2, but not by mutant proteins. Elimination of the second glycosylation site of AAT abolished LMAN1 dependent secretion. Co-immunoprecipitation experiment in MCFD2 KO cells suggested that AAT interaction with LMAN1 is independent of MCFD2. Furthermore, our results suggest that secretion of the Z variant, both monomers and polymers, is also LMAN1-dependent. Results provide direct evidence supporting that the LMAN1-MCFD2 complex is a cargo receptor for the ER-to-Golgi transport of AAT and that interactions of LMAN1 with an N-glycan of AAT is critical for this process. These results have implications in production of recombinant AAT and in developing treatments for AATD patients.

Succinylation is a newly discovered and multienzyme-regulated post-translational modification (PTM) that is associated with the initiation and progression of cancer. Currently, no systematic analyses on the role of succinylation regulators in tumors have been reported. In this study, we performed a comprehensive pan-cancer analysis on four well-known succinylation regulators (CPT1A, KAT2A, SIRT5, and SIRT7). We found that these regulators played specific and critical roles in the prognosis of clear cell renal cell carcinoma (ccRCC). We constructed a risk score (RS) based on two independent prognostic prediction factors, CPT1A and KAT2A, and subsequently developed a nomogram model containing the RS, which showed good accuracy in the prediction of overall survival (OS) in ccRCC patients. Furthermore, we used the similar expression pattern of four succinylation regulators according to consensus clustering analysis to divide the patients into three clusters that exhibited prominently different OS as well as clinicopathological characteristics. Differently expressed genes (DEGs) and pathway enrichment analyses of three clusters indicated that succinylation regulators might promote malignant progression of ccRCC by regulating the infiltration of immune cells and RNA N6-methyladenosine (m6A) methylation. Importantly, our data suggest that CPT1A and SIRT5 might up-regulate and down-regulate the expression of LRPPRC and EIF3B, respectively. Our study systematically analyzed the prognostic predictive values of four succinylation regulators and revealed their potential mechanisms in ccRCC aggressiveness. These data provide new insight into the understanding of succinylation modification and present clinical evidence for its role in ccRCC treatments.

Patients with non-small cell lung cancer (NSCLC) treated with EGFR-tyrosine kinase inhibitors (TKIs) ultimately develop drug resistance and metastasis. Therefore, there is a need to identify the underlying mechanisms of resistance to EGFR-TKIs. In the present study, colony formation and MTT assays were performed to investigate cell viability following treatment with icotinib. Gene Expression Omnibus datasets were used to identify genes associated with resistance. Wound healing and Transwell assays were used to detect cell migration and invasion with icotinib treatment and integrin α5-knockdown. The expression levels of integrin α5 and downstream genes were detected using western blotting. Stable icotinib-resistant (IcoR) cell lines (827/IcoR and PC9/IcoR) were established that showed enhanced malignant properties compared with parental cells (HCC827 and PC9). Furthermore, the resistant cell lines were resistant to icotinib in terms of proliferation, migration and invasion. The enrichment of function and signaling pathways analysis showed that integrin α5-upregulation was associated with the development of icotinib resistance. The knockdown of integrin α5 attenuated the migration and invasion capability of the resistant cells. Moreover, a combination of icotinib and integrin α5 siRNA significantly inhibited migration and partly restored icotinib sensitivity in IcoR cells. The expression levels of phosphorylated (p)-focal adhesion kinase (FAK), p-STAT3 and p-AKT decreased after knockdown of integrin α5, suggesting that FAK/STAT3/AKT signaling had a notable effect on the resistant cells. The present study revealed that the integrin α5/FAK/STAT3/AKT signaling pathway promoted icotinib resistance and malignancy in IcoR NSCLC cells. This signaling pathway may provide promising targets against acquired resistance to EGFR-TKI in patients with NSCLC.

Background: Breast cancer is one of the most frequent malignant tumors worldwide, with 1.67 million newly-diagnosed cases and 522,000 deaths each year. Therefore, seeking the novel biomarkers and therapeutic targets that contribute to postoperative recurrence and metastasis in patients with breast cancer is emerging and facilitates the development of innovative therapeutics. Methods: Retrieving the dataset of patients with hormone receptor (HR)-positive breast cancers from Gene Expression Omnibus (GEO) and collecting the data from the patients with HR-positive breast cancers enrolled in the First Affiliated Hospital of China Medical University are so as to identify the miRNAs associated with metastasis and distant metastasis-free survival (DMFS). Then MTT and Transwell migration assays were used to validate the effect of miRNAs on cell proliferation and migration of estrogen receptor-positive breast cancer T47D and MCF7 cells in vitro, respectively. Results: From GSE59829 dataset, the miRNA expression levels of miR-891a-5p, miR-383-5p and miR-1295a were significantly downregulated while the levels of miR-128-3p, miR-661 and miR-296-3p were significantly upregulated in breast cancers from patients with metastasis as compared to the matched non-metastatic group. Moreover, low expression levels of miR-891a-5p, miR-383-5p and miR-1295a or high expression levels of miR-128-3p, miR-661 and miR-296-3p were respectively associated with low DMFS in patients with breast cancer. Our clinical cohort study supported that the levels of miR-891a-5p, miR-383-5p and miR-1295a were significantly lower in breast cancers from the metastasis group when compared with non-metastatic group. However, there is no significant difference with regard to the levels of miR-128-3p, miR-661 and miR-296-3p in breast cancer between these two groups. Moreover, low expression levels of miR-891a-5p and miR-383-5p but not miR-1295a in breast cancer were significantly associated with low DMFS in patients, implying that the expression of miR-891a-5p and miR-383-5p were the potential prognosis markers for metastatic human breast cancers. Further investigation disclosed that miR-891a-5p but not miR-383-5p restrained both proliferation and migration of T47D and MCF7 cells. In silico analysis of miRNAs target gene through online computational algorithms revealed that A Disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) is the downstream target for miR-891a-5p. Further study confirmed that miR-891a-5p impeded ADAM10 expression by directly binding to its 3'UTR, leading to the inhibition of breast cancer cells proliferation and migration. Moreover, silencing ADAM10 inhibited T47D and MCF7 cells growth and migration. Conclusion: miR-891a-5p is the vital prognostic marker for HR-positive breast cancer. In addition, miR-891a-5p and miR-383-5p are the potential targets for HR-positive breast cancer therapeutics.

This study aimed to explore the specific pathogenesis of lncRNA MALAT1 promoting the invasion and metastasis of hepatocellular carcinoma (HCC) through peripheral blood vessels by regulating the expression of miRNA-613 molecule.

MicroRNAs (miRNAs) are known to be involved in the development and progression of pancreatic cancer (PC). In this study, the prognostic significance and mechanistic role of microRNA-569 in PC were explored. Quantitative real-time PCR was used to detect the expression of microRNA-569 in PC tissues and cell lines. Scratch test and Transwell assay were conducted to detect migration and invasion ability. The xenograft nude mice model was used to determine tumor metastasis in vivo. The direct targets of microRNA-569 were determined by using bioinformatics analysis and a dual-luciferase reporter assay. The expression level of microRNA-569 was down-regulated in PC patients with a poor prognosis. In vitro and in vivo experiments indicated that over-expression of microRNA-569 inhibited the migration and invasion of PC cells. MicroRNA-569 negatively regulated NUSAP1 by directly binding its 3'-untranslated region. Further mechanism research implied that the ZEB1 pathway was involved in microRNA-569/NUSAP1 mediation of the biological behaviors in PC. These data demonstrated that microRNA-569 may exert a tumor-suppressing effect in PC and maybe a potential therapeutic target for PC patients.

Colorectal cancers (CRCs) with the BRAF V600E mutation exhibit upregulation of programmed death ligand 1 (PD-L1) but fail to respond to immunotherapy targeting programmed cell death protein 1 (PD-1)/PD-L1. Recent studies have explored the intracellular functions of PD-L1. Here, we demonstrate that PD-L1 was highly expressed in both the cytoplasm and nucleus of BRAF-mutated CRC tumor cells and tissues. Nuclear PD-L1 (nPD-L1) promoted the growth of tumor cells both in vitro and in vivo. Mechanistic investigations revealed that PD-L1 translocation into the nucleus was facilitated by the binding of p-ERK. Further, nPD-L1 upregulated the expression of cell cycle regulator BUB1 via interactions with thyroid hormone receptor-associated protein 3 (THRAP3), thereby accelerating cell cycle progression and promoting cell proliferation. Moreover, BRAF V600E-mutated CRC cells exhibited upregulation of PD-L1 expression via the transcription factor LEF-1. These findings reveal a novel role of nPD-L1, which promotes cell cycle progression in an immune-independent manner in BRAF V600E-mutated CRC. Our study provides novel insight into the mechanisms underlying BRAF V600E-mutated CRC progression.

Aldehyde dehydrogenase 3A1 (ALDH3A1) is an NAD+-dependent enzyme that is closely related to tumor development. However, its role in non-small-cell lung cancer (NSCLC) has not been elucidated. This study aimed to clarify the mechanism of ALDH3A1 and identify potential therapeutic targets for NSCLC. Here, for the first time, we found that ALDH3A1 expression could be induced by a hypoxic environment in NSCLC. ALDH3A1 was highly expressed in NSCLC tissue, especially in some late-stage patients, and was associated with a poor prognosis. In mechanistic terms, ALDH3A1 enhances glycolysis and suppresses oxidative phosphorylation (OXPHOS) to promote cell proliferation by activating the HIF-1α/LDHA pathway in NSCLC. In addition, the results showed that ALDH3A1 was a target of β-elemene. ALDH3A1 can be downregulated by β-elemene to inhibit glycolysis and enhance OXPHOS, thus suppressing NSCLC proliferation in vitro and in vivo. In conclusion, hypoxia-induced ALDH3A1 is related to the energy metabolic status of tumors and the efficacy of β-elemene, providing a new theoretical basis for better clinical applications in NSCLC.

This study aimed to investigate the effect of NPTX1 on the prognosis of gastric cancer (GC), as well as the metastatic process in GC.

5-Florouracil (5-FU) is the basic agent used in the treatment of gastric cancer. Capecitabine, a prodrug of 5-FU, displays increased antitumor efficacy compared with 5-FU in the clinic. 5'-Deoxy-5-fluorouracil (5'-DFUR), the metabolite of capecitabine, is converted to 5-FU by the enzyme thymidine phosphorylase (TP), which is present at high concentrations in human tumors. In this study, we investigated the effect of interferon-α (IFN-α) on the sensitivity of gastric cancer cells to treatment with 5'-DFUR and its relationship with TP expression. Preincubation of gastric cancer cells with IFN-α enhanced 5'-DFUR-induced apoptosis via IFN-α-mediated upregulation of TP. The depletion of TP with small interfering RNA (siRNA) obviously inhibited IFN-α-induced upregulation of TP expression and thus prevented apoptosis induced by IFN-α and 5'-DFUR. Treatment with IFN-α and combined IFN-α and 5'-DFUR treatment were also associated with concomitant activation of ERK signaling. Treatment with the ERK inhibitor PD98059 or depletion of ERK with siRNA partially reversed IFN-α-induced upregulation of TP expression, thus partially preventing apoptosis induced by IFN-α and 5'-DFUR. Taken together, our study shows that IFN-α enhanced 5'-DFUR-induced apoptosis in gastric cancer cells by upregulation of TP expression, which is partially regulated by activation of ERK signaling.

The unicellular green alga, Chlamydomonas reinhardtii, is a classic model for studying flagella and biofuel. However, precise gene editing, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas9) system, is not widely used in this organism. Screening of random insertional mutant libraries by polymerase chain reaction provides an alternate strategy to obtain null mutants of individual gene. But building, screening, and maintaining such a library was time-consuming and expensive.