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Abstract The matrix metalloprotease A disintegrin and metalloprotease with thrombospondin motifs 1 (ADAMTS1) was reported to be involved in tumor progression in several cancer types, but its contributions appear discrepant. At present, the role of ADAMTS1 in oral squamous cell carcinoma (SCC; OSCC) remains unclear. Herein, The Cancer Genome Atlas (TCGA) database showed that ADAMTS1 transcripts were downregulated in head and neck SCC (HNSCC) tissues compared to normal tissues, but ADAMTS1 levels were correlated with poorer prognoses of HNSCC patients. In vitro, we observed that ADAMTS1 expression levels were correlated with the invasive abilities of four OSCC cell lines, HSC-3, SCC9, HSC-3M, and SAS. Knockdown of ADAMTS1 in OSCC cells led to a decrease and its overexpression led to an increase in cell-invasive abilities in vitro as well as tumor growth and lymph node (LN) metastasis in OSCC xenografts. Mechanistic investigations showed that the cyclic increase in ADAMTS1-L1 cell adhesion molecule (L1CAM) axis-mediated epidermal growth factor receptor (EGFR) activation led to exacerbation of the invasive abilities of OSCC cells via inducing epithelial-mesenchymal transition (EMT) progression. Clinical analyses revealed that ADAMTS1, L1CAM, and EGFR levels were all correlated with worse prognoses of HNSCC patients, and patients with ADAMTS1high/L1CAMhigh or EGFRhigh tumors had the shortest overall and disease-specific survival times. As to therapeutic aspects, we discovered that an edible plant-derived flavonoid, apigenin (API), drastically inhibited expression of the ADAMTS1-L1CAM-EGFR axis and reduced the ADAMTS1-triggered invasion and LN metastasis of OSCC cells in vitro and in vivo. Most importantly, API treatment significantly prolonged survival rates of xenograft mice with OSCC. In summary, ADAMTS1 may be a useful biomarker for predicting OSCC progression, and API potentially retarded OSCC progression by targeting the ADAMTS1-L1CAM-EGFR signaling pathway.

Abstract Pancreatic ductal adenocarcinoma (PDAC) tumours carry multiple gene mutations and respond poorly to treatments. There is currently an unmet need for drug carriers that can deliver multiple gene cargoes to target high solid tumour burden like PDAC. Here, we report a dual targeted extracellular vesicle (dtEV) carrying high loads of therapeutic RNA that effectively suppresses large PDAC tumours in mice. The EV surface contains a CD64 protein that has a tissue targeting peptide and a humanized monoclonal antibody. Cells sequentially transfected with plasmid DNAs encoding for the RNA and protein of interest by Transwell®-based asymmetric cell electroporation release abundant targeted EVs with high RNA loading. Together with a low dose chemotherapy drug, Gemcitabine, dtEVs suppress large orthotopic PANC-1 and patient derived xenograft tumours and metastasis in mice and extended animal survival. Our work presents a clinically accessible and scalable way to produce abundant EVs for delivering multiple gene cargoes to large solid tumours.

Abstract Background KH-type splicing regulatory protein (KHSRP, also called KSRP), a versatile RNA-binding protein, plays a critical role in various physiological and pathological conditions through modulating gene expressions at multiple levels. However, the role of KSRP in clear cell renal cell carcinoma (ccRCC) remains poorly understood. Methods KSRP expression was detected by a ccRCC tissue microarray and evaluated by an in silico analysis. Cell loss-of-function and gain-of-function, colony-formation, anoikis, and transwell assays, and an orthotopic bioluminescent xenograft model were conducted to determine the functional role of KRSP in ccRCC progression. Micro (mi)RNA and complementary (c)DNA microarrays were used to identify downstream targets of KSRP. Western blotting, quantitative real-time polymerase chain reaction, and promoter- and 3-untranslated region (3'UTR)-luciferase reporter assays were employed to validate the underlying mechanisms of KSRP which aggravate progression of ccRCC. Results Our results showed that dysregulated high levels of KSRP were correlated with advanced clinical stages, larger tumor sizes, recurrence, and poor prognoses of ccRCC. Neural precursor cell-expressed developmentally downregulated 4 like (NEDD4L) was identified as a novel target of KSRP, which can reverse the protumorigenic and prometastatic characteristics as well as epithelial-mesenchymal transition (EMT) promotion by KSRP in vitro and in vivo. Molecular studies revealed that KSRP can decrease NEDD4L messenger (m)RNA stability via inducing mir-629-5p upregulation and directly targeting the AU-rich elements (AREs) of the 3’UTR. Moreover, KSRP was shown to transcriptionally suppress NEDD4L via inducing the transcriptional repressor, Wilm's tumor 1 (WT1). In the clinic, ccRCC samples revealed a positive correlation between KSRP and mesenchymal-related genes, and patients expressing high KSRP and low NEDD4L had the worst prognoses. Conclusion The current findings unveil novel mechanisms of KSRP which promote malignant progression of ccRCC through transcriptional inhibition and post-transcriptional destabilization of NEDD4L transcripts. Targeting KSRP and its pathways may be a novel pharmaceutical intervention for ccRCC.

Abstract Renal cell carcinoma (RCC) is one of the most lethal genitourinary malignancies with poor prognoses, since it is largely resistant to chemotherapy, radiotherapy, and targeted therapy. The persistence of cancer stem cells (CSCs) is the major cause of treatment failure with RCC. Recent evidence showed that dopamine receptor D2 (DRD2)-targeting antipsychotic drugs such as penfluridol exert oncostatic effects on several cancer types, but the effect of penfluridol on RCC remains unknown. Here, we uncovered penfluridol suppressed in vitro cell growth and in vivo tumorigenicity of various RCC cell lines (Caki-1, 786-O, A498, and ACHN) and enhanced the Sutent (sunitinib)-triggered growth inhibition on clear cell (cc)RCC cell lines. Mechanistically, upregulation of endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) was critical for autophagy-mediated apoptosis induced by penfluridol. Transcriptional inhibition of OCT4 and Nanog via inhibiting GLI1 was important for penfluridol-induced stemness and proliferation inhibition. The anticancer activities of penfluridol on ccRCC partially occurred through DRD2. In clinical ccRCC specimens, positive correlations of DRD2 with GLI1, OCT4, and Nanog were observed and their expressions were correlated with worse prognoses. Summarizing, DRD2 antagonists such as penfluridol induce UPR signaling and suppress the GLI1/OCT4/Nanog axis in ccRCC cells to reduce their growth through inducing autophagy-mediated apoptosis and stemness inhibition. These drugs can be repurposed as potential agents to treat ccRCC patients.

Non-small-cell lung cancer (NSCLC) is a typical inflammation-associated cancer, and lung adenocarcinoma (LUAD) is the most common pathological subtype. Epidermal growth factor (EGF) receptor (EGFR) mutations are the most common driver mutations of LUAD, and they have been identified as important therapeutic targets by EGFR-tyrosine kinase inhibitors (TKIs). The proinflammatory cytokine, interleukin (IL)-17A, and IL-17A-producing cells were reported to be elevated in the tumor microenvironment and peripheral blood of NSCLC patients and to be correlated with tumor progression and poor prognoses. However, the pathophysiological role of IL-17A in NSCLC remains unclear, although some studies suggested its involvement in cancer cell invasion and metastasis. Herein, we observed that expressions of IL-17A and its receptor, IL-17 receptor C (IL-17RC), were elevated in LUAD tissues and were correlated with poor survival in different lung cancer cohorts. In LUAD cells with mutant EGFR, the IL-17A/IL-17RC axis was shown to enhance phosphorylation of EGFR and Met, thereby promoting proliferation and resistance to EGFR-TKIs such as afatinib. In LUAD cells with wild-type (WT) EGFR, we found that the IL-17A/IL-17RC axis enhanced EGF-induced EGFR activation and cell proliferation through causing impairment of EGF-induced EGFR lysosomal degradation. Collectively, our results indicated diverse impacts of the IL-17A/IL-17RC axis on EGFR activation in LUAD cells with WT and mutant EGFR and suggested that developing therapeutic strategies against IL-17A/IL-17RC would be valuable for LUAD treatment.

Abstract Background Due to the difficulties in early diagnosing and treating hepatocellular carcinoma (HCC), prognoses for patients remained poor in the past decade. In this study, we established a screening model to discover novel prognostic biomarkers in HCC patients. Methods Candidate biomarkers were screened by liquid chromatography with tandem mass spectrometry (LC-MS/MS) analyses of five HCC normal (N)/tumor (T) paired tissues and preliminarily verified them through several in silico database analyses. Expression levels and functional roles of candidate biomarkers were respectively evaluated by immunohistochemical staining in N/T paired tissue (n = 120) and MTS, colony formation, and transwell migration/invasion assays in HCC cell lines. Associations of clinicopathological features and prognoses with candidate biomarkers in HCC patients were analyzed from GEO and TCGA datasets and our recruited cohort. Results We found that the transmembrane P24 trafficking protein 9 (TMED9) protein was elevated in HCC tissues according to a global proteomic analysis. Higher messenger (m)RNA and protein levels of TMED9 were observed in HCC tissues compared to normal liver tissues or pre-neoplastic lesions. The TMED9 mRNA expression level was significantly associated with an advanced stage and a poor prognosis of overall survival (OS, p = 0.00084) in HCC patients. Moreover, the TMED9 protein expression level was positively correlated with vascular invasion (p = 0.026), OS (p = 0.044), and disease-free survival (p = 0.015) in our recruited Taiwanese cohort. In vitro, manipulation of TMED9 expression in HCC cells significantly affected cell migratory, invasive, proliferative, and colony-forming abilities. Conclusions Ours is the first work to identify an oncogenic role of TMED9 in HCC cells and may provide insights into the application of TMED9 as a novel predictor of clinical outcomes and a potential therapeutic target in patients with HCC.

Sparc/osteonectin, cwcv, and kazal-like domains proteoglycan 1 (SPOCK1) has been reported to play an oncogenic role in certain cancer types; however, the role of SPOCK1 in the progression of clear cell renal cell carcinoma (ccRCC) remains elusive. Here, higher SPOCK1 transcript and protein levels were observed in ccRCC tissues compared to normal tissues and correlated with advanced clinical stages, larger tumor sizes, and lymph node and distal metastases. Knockdown and overexpression of SPOCK1 in ccRCC cells led to decreased and increased cell clonogenic and migratory/invasive abilities in vitro as well as lower and higher tumor growth and invasion in vivo, respectively. Mechanistically, the gene set enrichment analysis (GSEA) database was used to identify the gene set of epithelial-to-mesenchymal transition (EMT) pathways enriched in ccRCC samples with high SPOCK1 expression. Further mechanistic investigations revealed that SPOCK1 triggered the Snail/Slug–matrix metalloproteinase (MMP)-2 axis to promote EMT and cell motility. Clinical ccRCC samples revealed SPOCK1 to be an independent prognostic factor for overall survival (OS), and positive correlations of SPOCK1 with MMP-2 and mesenchymal-related gene expression levels were found. We observed that patients with SPOCK1high/MMP2high tumors had the shortest OS times compared to others. In conclusion, our findings reveal that SPOCK1 can serve as a useful biomarker for predicting ccRCC progression and prognosis, and as a promising target for treating ccRCC.

Long noncoding (lnc)RNAs are reported to be key regulators of tumor progression, including hepatocellular carcinoma (HCC). The lncRNA long intergenic noncoding RNA 00673 (LINC00673) was indicated to play an important role in HCC progression, but the impacts of genetic variants (single-nucleotide polymorphisms, SNPs) of LINC00673 on HCC remain unclear. A TaqMan allelic discrimination assay was performed to analyze the genotypes of three tagging SNPs, viz., rs9914618 G > A, rs6501551 A > G, and rs11655237 C > T, of LINC00673 in 783 HCC patients and 1197 healthy subjects. Associations of functional SNPs of LINC00673 with HCC susceptibility and clinicopathologic variables were analyzed by logistic regression models. After stratification by confounding factor, we observed that elderly patients (≥60 years) with the LINC00673 rs9914618 A allele had an increased risk of developing HCC under a codominant model (p = 0.025) and dominant model (p = 0.047). Moreover, elderly patients carrying the GA + AA genotype of rs9914618 exhibited a higher risk of having lymph node metastasis compared to those who were homozygous for the major allele (p = 0.013). Genotype screening of rs9914618 in HCC cell lines showed that cells carrying the AA genotype expressed higher LINC00673 levels compared to the cells carrying the GG genotype. Further analyses of clinical datasets from the Cancer Genome Atlas (TCGA) showed that LINC00673 expressions were upregulated in HCC tissues compared to normal tissues, and were correlated with advanced clinical stages and poorer prognoses. In conclusions, our results suggested that the LINC00673 rs9914618 polymorphism may be a promising HCC biomarker, especially in elderly populations.

Oral squamous cell carcinoma (OSCC) is the most common malignant tumor of the oral cavity, and long non-coding (lnc)RNA of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was recently reported to play a crucial role in OSCC development and progression. However, potential effects of genetic variants of MALAT1 on the development of OSCC are still unclear. Herein, we performed a case-control study in 1350 patients with OSCC and 1199 healthy controls to evaluate the association between functional single-nucleotide polymorphisms (SNPs) of MALAT1 and OSCC susceptibility, as well as its clinicopathologic characteristics. A TaqMan allelic discrimination assay was used to genotype four tagging SNPs, viz., rs3200401 C>T, rs619586 A>G, rs1194338 C>A, and rs7927113 G>A, and results showed that the MALAT1 rs3200401 T allele had a lower risk of OSCC (adjusted odds ratio (AOR): 0.779, 95% confidence interval (CI): 0.632~0.960, p=0.019) and a higher risk of developing moderately (grade II)/poorly (grade III) differentiated OSCC (AOR: 1.508-fold, 95% CI: 1.049~2.169, p=0.027) under a dominant model. According to environmental carcinogen exposure, patients with a betel quid-chewing habit who carried the T allele of rs3200401 more easily developed high-grade (II/III) OSCC (AOR: 1.588, 95% CI: 1.055~2.390, p=0.027), and patients with the same genotype but who did not chew betel quid had a lower risk of developing lymph node metastasis (AOR: 0.437, 95% CI: 0.255~0.749, p=0.003). In addition to rs3200401, the rs619586 AG/GG genotype was associated with increased risks of developing advanced stages (III+IV) and larger tumor sizes (>T2) compared to the AA genotype, especially in the subgroup of betel quid chewers. Furthermore, analyses of clinical datasets revealed that the MALAT1 expression level was upregulated in OSCC compared to normal tissues, especially in the betel quid-chewing population. These results indicated involvement of MALAT1 SNPs rs3200401 and rs619586 in the development of OSCC and support the interaction between MALAT1 gene polymorphisms and the environmental carcinogen as a predisposing factor for OSCC progression.

Abstract Background Chemotherapy is the main treatment for acute myeloid leukemia (AML), but the cure rates for AML patients remain low, and the notorious adverse effects of chemotherapeutic drugs drastically reduce the life quality of patients. Penfluridol, a long-acting oral antipsychotic drug, has an outstanding safety record and exerts oncostatic effects on various solid tumors. Until now, the effect of penfluridol on AML remains unknown. Methods AML cell lines harboring wild-type (WT) Fms-like tyrosine kinase 3 (FLT3) and internal tandem duplication (ITD)-mutated FLT3 were used to evaluate the cytotoxic effects of penfluridol by an MTS assay. A flow cytometric analysis and immunofluorescence staining were employed to determine the cell-death phenotype, cell cycle profile, and reactive oxygen species (ROS) and acidic vesicular organelle (AVO) formation. Western blotting and chemical inhibitors were used to explore the underlying mechanisms involved in penfluridol-mediated cell death. Results We observed that penfluridol concentration-dependently suppressed the cell viability of AML cells with FLT3-WT (HL-60 and U937) and FLT3-ITD (MV4–11). We found that penfluridol treatment not only induced apoptosis as evidenced by increases of nuclear fragmentation, the sub-G1 populations, poly (ADP ribose) polymerase (PARP) cleavage, and caspase-3 activation, but also triggered autophagic responses, such as the light chain 3 (LC3) turnover and AVO formation. Interestingly, blocking autophagy by the pharmacological inhibitors, 3-methyladenine and chloroquine, dramatically enhanced penfluridol-induced apoptosis, indicating the cytoprotective role of autophagy in penfluridol-treated AML cells. Mechanistically, penfluridol-induced apoptosis occurred through activating protein phosphatase 2A (PP2A) to suppress Akt and mitogen-activated protein kinase (MAPK) activities. Moreover, penfluridol’s augmentation of intracellular ROS levels was critical for the penfluridol-induced autophagic response. In the clinic, we observed that patients with AML expressing high PP2A had favorable prognoses. Conclusions These findings provide a rationale for penfluridol being used as a PP2A activator for AML treatment, and the combination of penfluridol with an autophagy inhibitor may be a novel strategy for AML harboring FLT3-WT and FLT3-ITD.

Abstract Background Testicular torsion is an urological emergency that may lead to infertility due to ischemic injury. Promptly surgical correction by orchiopexy is the only way to avoid infertility and no effective treatment for restoration of spermatogenesis. We previously reported that mesenchymal stem cells (MSCs), through local injection upon testicular torsion-detorsion, restored the spermatogenesis without differentiation into sperm. In this study, molecular mechanisms of MSCs in regulating germ cell activity induced by testicular torsion-detorsion were investigated. Methods Sixteen male Sprague-Dawley rats 6–8 weeks old received left testis 720° torsion for 3 h followed by detorsion with or without MSCs. Right inguinal skin incision without testicular torsion served as control. MSCs with 3 × 104 cells were locally injected into left testis 30 min before detorsion. Three days after the surgery, orchiectomy was executed and the testis, epididymis, and sperm were separated to each other. Functional assessments on sperm included counting sperm amount and sperm motility, staining F-actin, and quantifying adenosine triphosphate (ATP) content. The hallmarks of glycogenesis and glycolysis in each tissue segment were measured by Western blot. Results Testicular torsion-detorsion significantly decreased the amount of sperm, inhibited the motility, declined the F-actin expression, and reduced the content of ATP in sperm. Local injection of MSCs improved sperm function, particularly in sperm motility. With MSCs, ATP content and F-actin were preserved after testicular torsion-detorsion. MSCs significantly reversed the imbalance of glycolysis in sperm and testis induced by testicular torsion-detorsion, as evidenced by increasing the expression of phosphoglycerate kinase 2 and glyceraldehyde-3-phosphate dehydrogenase-spermatogenic, activating Akt, and increasing glycogen synthase kinase 3 (GSK3), which led to the increase in glycolysis cascades and ATP production. Human stem cell factor contributed the activation of Akt/GSK3 axis when sperm suffered from testicular torsion-detorsion-induced germ cell injury. Conclusions Local injection of MSCs into a testis damaged by testicular torsion-detorsion restores sperm function mainly through the improvement of sperm motility and energy. MSCs reversed the imbalance of glycogenesis and glycolysis in sperm by regulating Akt/GSK3 axis. Thus, MSCs may potentially rescue torsion-detorsion-induced infertility via local injection.

Abstract Background Prostate cancer (PCa) is considered one of the most prevalent malignancy globally, and metastasis is a major cause of death. Apigenin (API) is a dietary flavonoid which exerts an antimetastatic effect in various cancer types. Sparc/osteonectin, cwcv, and kazal-like domains proteoglycan 1 (SPOCK1) is a crucial modulator of tumor growth and metastasis in cancers. However, the role and underlying regulatory mechanisms of SPOCK1 in the API-mediated antimetastatic effects of PCa remain unclear. Methods MTS, colony formation, wound-healing, and transwell assays were conducted to evaluate the effects of API on PCa cell proliferative, migratory, and invasive potentials. In vivo orthotopic bioluminescent xenograft model were employed to determine antitumor activity of API. PCa cells were transfected with either Snail-, Slug-, SPOCK1-overexpressing vector, or small hairpin (sh)SPOCK1 to determine the invasive abilities and expression levels of SPOCK1 and epithelial-to-mesenchymal transition (EMT) biomarkers in response to API treatment. Immunohistochemical (IHC) assays were carried out to evaluate the expression level of SPOCK1 in PCa xenografts and a PCa tissue array. Associations of SPOCK1 expression with clinicopathological features and prognoses of patients with PCa were analyzed by GEO or TCGA RNA-sequencing data. Results API significantly suppressed in vitro PCa cell proliferation, migration, and invasion and inhibited in vivo PCa tumor growth and metastasis. Moreover, survival times of animals were also prolonged after API treatment. Mechanistic studies revealed that API treatment resulted in downregulation of SPOCK1, which was accompanied by reduced expressions of mesenchymal markers and subsequent attenuation of invasive abilities of PCa cells. Overexpression of SPOCK1 in PCa xenografts resulted in significant promotion of tumor progression and relieved the anticancer activities induced by API, whereas knockdown of SPOCK1 had opposite effects. In clinical, SPOCK1 levels were higher in tumor tissues compared to non-tumor tissues, which was also significantly correlated with shorter disease-free survival in PCa patients. Conclusions Levels of SPOCK1 increase with the progression of human PCa which suggests that SPOCK1 may act as a prognostic marker or therapeutic target for patients with PCa. Suppression of SPOCK1-mediated EMT signaling contributes to the antiproliferative and antimetastatic activities of API in vitro and in vivo.

Energy metabolism is the basis for cell growth, and cancer cells in particular, are more energy-dependent cells because of rapid cell proliferation. Previously, we found that penfluridol, an antipsychotic drug, has the ability to trigger cell growth inhibition of lung cancer cells via inducing ATP energy deprivation. The toxic effect of penfluridol is related to energy metabolism, but the underlying mechanisms remain unclear. Herein, we discovered that treatment of A549 and HCC827 lung cancer cells with penfluridol caused a decrease in the total amount of ATP, especially in A549 cells. An Agilent Seahorse ATP real-time rate assay revealed that ATP production rates from mitochondrial respiration and glycolysis were, respectively, decreased and increased after penfluridol treatment. Moreover, the amount and membrane integrity of mitochondria decreased, but glycolysis-related proteins increased after penfluridol treatment. Furthermore, we observed that suppression of glycolysis by reducing glucose supplementation or using 2-deoxy-D-glucose (2DG) synergistically enhanced the inhibitory effect of penfluridol on cancer cell growth and the total amount of mitochondria. A mechanistic study showed that the penfluridol-mediated energy reduction was due to inhibition of critical regulators of mitochondrial biogenesis, the sirtuin 1 (SIRT1)/peroxisome-proliferator-activated receptor co-activator-1α (PGC-1α) axis. Upregulation of the SIRT1/PGC-1α axis reversed the inhibitory effect of penfluridol on mitochondrial biogenesis and cell viability. Clinical lung cancer samples revealed a positive correlation between PGC-1α (PPARGC1A) and SIRT1 expression. In an orthotopic lung cancer mouse model, the anticancer activities of penfluridol, including growth and metastasis inhibition, were also enhanced by combined treatment with 2DG. Our study results strongly support that a combination of repurposing penfluridol and a glycolysis inhibitor would be a good strategy for enhancing the anticancer activities of penfluridol in lung cancer.

Background/Aims: Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) therapy is a clinical option for non-small cell lung cancer (NSCLC) harboring activating EGFR mutations or for cancer with wild-type (WT) EGFR when chemotherapy has failed. MET receptor activation or MET gene amplification was reported to be a major mechanism of acquired resistance to EGFR-TKI therapy in NSCLC cells. Leukocyte cell-derived chemotaxin 2 (LECT2) is a multifunctional cytokine that was shown to suppress metastasis of hepatocellular carcinoma via inhibiting MET activity. Until now, the biological function responsible for LECT2’s action in human NSCLC remains unclear. Methods: LECT2-knockout (KO) mice and NOD/SCID/IL2rgnull (NSG) mice were respectively used to investigate the effects of LECT2 on the tumorigenicity and metastasis of murine (Lewis lung carcinoma, LLC) and human (HCC827) lung cancer cells. The effect of LECT2 on in vitro cell proliferation was evaluated, using MTS and colony formation assays. The effect of LECT2 on cell motility was evaluated using transwell migration and invasion assays. An enzyme-linked immunosorbent assay was performed to detect secreted LECT2 in plasma and media. Co-immunoprecipitation and Western blot assays were used to investigate the underlying mechanisms of LECT2 in NSCLC cells. Results: Compared to WT mice, mice with LECT2 deletion exhibited enhanced growth and metastasis of LLC cells, and survival times decreased in LLC-implanted mice. Overexpression of LECT2 in orthotopic human HCC827 xenografts in NSG mice resulted in significant inhibition of tumor growth and metastasis. In vitro, overexpression of LECT2 or treatment with a recombinant LECT2 protein impaired the colony-forming ability and motility of NSCLC cells (HCC827 and PC9) harboring high levels of activated EGFR and MET. Mechanistic investigations found that LECT2 bound to MET and EGFR to antagonize their activation and further suppress their common downstream pathways: phosphatidylinositol 3-kinase/Akt and extracellular signal-regulated kinase. Conclusion: EGFR-MET signaling is critical for aggressive behaviors of NSCLC and is recognized as a therapeutic target for NSCLC especially for patients with acquired resistance to EGFR-TKI therapy. Our findings demonstrate, for the first time, that LECT2 functions as a suppressor of the progression of NSCLC by targeting EGFR-MET signaling.

Abstract Background Metastasis rather than the primary cancer determines the survival of cancer patients. Activation of Akt plays a critical role in the epithelial-to-mesenchymal transition (EMT), the initial step in lung cancer metastasis. Apigenin (API), a flavonoid with a potent Akt-inhibitory effect, shows oncostatic activities in various cancers. However, the effects of API on metastasis of non-small cell lung cancer (NSCLC) remain unclear. Methods NSCLC cell lines with different epidermal growth factor receptor (EGFR) statuses and in vivo orthotopic bioluminescent xenograft model were employed to determine antitumor activity of API. Western blot and genetic knockdown by shRNA or genetic overexpression by DNA plasmids were performed to explore the underlying mechanisms. The Cancer Genome Atlas (TCGA) database was used to investigate the prognosis of API-targeted genes. Results API was demonstrated to inhibit the migration/invasion of NSCLC cells harboring different EGFR statuses via suppressing the Snail/Slug-mediated EMT. Mechanistic investigations showed that CD26/dipeptidyl peptidase IV (DPPIV) was downregulated by API following suppressive interplay of Akt and Snail/Slug signaling to modulate the EMT and the invasive ability of NSCLC cells. CD26 expression was positively correlated with the invasive abilities of NSCLC cells and a worse prognosis of lung cancer patients. Furthermore, we observed that patients with CD26high/Akthigh tumors had the shortest recurrence-free survival times. In vivo, API drastically reduced the growth and metastasis of A549 xenografts through targeting CD26. Conclusions CD26 may be a useful biomarker for predicting NSCLC progression. API effectively suppressed lung cancer progression by targeting the CD26-Akt-Snail/Slug signaling pathway.

Abstract Background The epidermal growth factor receptor (EGFR) plays important roles in cell survival, growth, differentiation, and tumorigenesis. Dysregulation of the EGFR is a common mechanism in cancer progression especially in non-small cell lung cancer (NSCLC). Main body Suppression of the EGFR-mediated signaling pathway is used in cancer treatment. Furthermore, reactive oxygen species (ROS)-induced oxidative stress from mitochondrial dysfunction or NADPH oxidase (NOX) overactivation and ectopic expression of antioxidative enzymes were also indicated to be involved in EGFR-mediated tumor progression (proliferation, differentiation, migration, and invasion) and drug resistance (EGFR tyrosine kinase inhibitor (TKI)). The products of NOX, superoxide and hydrogen peroxide, are considered to be major types of ROS. ROS are not only toxic materials to cells but also signaling regulators of tumor progression. Oxidation of both the EGFR and downstream phosphatases by ROS enhances EGFR-mediated signaling and promotes tumor progression. This review primarily focuses on the recent literature with respect to the roles of the EGFR and ROS and correlations between ROS and the EGFR in tumor progression and EGFR TKI resistance. Short conclusion The evidence discussed in this article can serve as a basis for basic and clinical research to understand how to modulate ROS levels to control the development and drug resistance of cancers.

Background: Oral squamous cell carcinoma (OSCC) has a high prevalence and predicted global mortality rate of 67.1%, necessitating better therapeutic strategies. Moreover, the recurrence and resistance of OSCC after chemo/radioresistance remains a major bottleneck for its effective treatment. Molecular targeting is one of the new therapeutic approaches to target cancer. Among a plethora of targetable signaling molecules, PDK1 is currently rising as a potential target for cancer therapy. Its aberrant expression in many malignancies is observed associated with glycolytic re-programming and chemo/radioresistance. Methods: Furthermore, to better understand the role of PDK1 in OSCC, we analyzed tissue samples from 62 patients with OSCC for PDK1 expression. Combining in silico and in vitro analysis approaches, we determined the important association between PDK1/CD47/LDHA expression in OSCC. Next, we analyzed the effect of PDK1 expression and its connection with OSCC orosphere generation and maintenance, as well as the effect of the combination of the PDK1 inhibitor BX795, cisplatin and radiotherapy in targeting it. Results: Immunohistochemical analysis revealed that higher PDK1 expression is associated with a poor prognosis in OSCC. The immunoprecipitation assay indicated PDK1/CD47 binding. PDK1 ligation significantly impaired OSCC orosphere formation and downregulated Sox2, Oct4, and CD133 expression. The combination of BX795 and cisplatin markedly reduced in OSCC cell’s epithelial-mesenchymal transition, implying its synergistic effect. p-PDK1, CD47, Akt, PFKP, PDK3 and LDHA protein expression were significantly reduced, with the strongest inhibition in the combination group. Chemo/radiotherapy together with abrogation of PDK1 inhibits the oncogenic (Akt/CD47) and glycolytic (LDHA/PFKP/PDK3) signaling and, enhanced or sensitizes OSCC to the anticancer drug effect through inducing apoptosis and DNA damage together with metabolic reprogramming. Conclusions: Therefore, the results from our current study may serve as a basis for developing new therapeutic strategies against chemo/radioresistant OSCC.

Hepatocellular carcinoma (HCC) accounts for the majority of primary liver cancers, which is the second most lethal tumor worldwide. Epigenetic deregulation is a common trait observed in HCC. Recently, increasing evidence suggested that the G9a histone methyltransferase might be a novel regulator of HCC development. However, several HCC cell lines were recently noted to have HeLa cell contamination or to have been derived from non-hepatocellular origin, suggesting that functional validation of G9a in proper HCC models is still required. Herein, we first confirmed that higher G9a messenger RNA and protein expression levels were correlated with poor overall survival (OS) and disease-free survival (DFS) rates of HCC patients from The Cancer Genome Atlas (TCGA) dataset and our recruited HCC cohort. In an in vitro functional evaluation of HCC cells, HCC36 (hepatitis B virus-positive (HBV+) and Mahlavu (HBV−)) cells showed that G9a participated in promoting cell proliferation, colony formation, and migration/invasion abilities. Moreover, orthotopic inoculation of G9a-depleted Mahlavu cells in NOD-SCID mice also resulted in a significantly decreased tumor burden compared to the control group. Furthermore, after surveying microRNA (miRNA; miR) prediction databases, we identified the liver-specific miR-122 as a G9a-targeting miRNA. In various HCC cell lines, we observed that miR-122 expression levels tended to be inversely correlated to G9a expression levels. In clinical HCC specimens, a significant inverse correlation of miR-122 and G9a mRNA expression levels was also observed. Functionally, the colony formation and invasive ability were attenuated in miR-122-overexpressing HCC cells. HCC patients with low miR-122 and high G9a expression levels had the worst OS and DFS rates compared to others. Together, our results confirmed the importance of altered G9a expression during HCC progression and discovered that a novel liver-specific miR-122-G9a regulatory axis exists.

Abstract Carbonic anhydrase IX (CA9) expression level has been considered as a poor prognostic factor in hepatocellular carcinoma (HCC) patients. However, the judging criteria of CA9 level is hard to define for potential clinical applications. Unlike CA9 expression level, CA9 polymorphism is poorly documented in HCC. Here, we found that people carry A allele at CA9 rs1048638, a 3′UTR SNP, has higher risk of HCC. rs1048638-CA correlates with advanced stages, larger tumor sizes, more vascular invasion, and shorter survival of HCC patients. A allele at CA9 rs1048638 impairs miR-34a, a tumor suppressor miRNA in HCC, binding to CA9 3′UTR and desensitizes CA9 mRNA to miR-34a-dependent RNA degradation. CA9 expression levels were also correlated with miR-34a levels and rs1048638 genotypes in HCC patients. rs1048638 influences HCC risk and progression through effects on miR-34a-targeted CA9 expression in HCC. In conclusion, genetic variations of the CA9 3′UTR play important roles in regulating CA9 expression and cancer progression, which is a novel determinant and target for HCC metastasis and prognosis.

Non-small cell lung cancer (NSCLC) is a typical inflammation-associated cancer, and lung adenocarcinoma (LUAD) is the most common histopathological subtype. Epidermal growth factor receptor (EGFR) mutations are the most common driver mutations of LUAD, and they have been identified as important therapeutic targets by EGFR tyrosine kinase inhibitors. Interleukin (IL)-17A secreted by T-helper 17 lymphocytes is a proinflammatory cytokine that plays an important role in cancer pathogenesis. The present study was designed to investigate the possible associations among IL-17A genetic polymorphisms, EGFR mutation status, and the clinicopathologic development of LUAD in a Taiwanese population. Our study population consisted of 277 LUAD patients harboring the wild-type (WT) EGFR or a mutant (MT) EGFR. Four single-nucleotide polymorphisms (SNPs) of IL-17A in the peripheral blood, including rs8193036(C > T), rs8193037(G > A), rs2275913(G > A), and rs3748067(C > T) loci, were genotyped using a TaqMan allelic discrimination assay. Our results showed that none of these IL-17A SNPs were correlated with the risk of developing mutant EGFR. However, patients with a smoking habit who carried the GA genotype of IL-17A rs8193037 had a significantly lower susceptibility to EGFR mutations (adjusted odds ratio (AOR): 0.225; 95% confidence interval (CI): 0.056~0.900, p = 0.035). Moreover, compared to individuals carrying the CC genotype of rs8193036 at IL-17A, T-allele carriers (CT + TT) were at higher risk of developing more-advanced stages (stage III or IV; p = 0.020). In the WT EGFR subgroup analysis, IL-17A rs8193036 T-allele carriers had higher risks of developing an advanced tumor stage (p = 0.016) and lymphatic invasion (p = 0.049). Further analyses of clinical datasets revealed correlations of IL-17 receptor A (IL-17RA) and IL-17RC expressions with a poor prognosis of LUAD patients with a smoking history or with higher levels of tumor-infiltrating lymphocytes. In conclusion, our results suggested that two functional promoter polymorphisms of IL-17A, i.e., rs8193036 and rs8193037, were associated with the EGFR mutation status and progression in LUAD patients, indicating that these two genetic variants might act as possible markers for predicting patients’ clinical prognoses.

The authors wish to make the following corrections to this paper [...]

Lung adenocarcinoma (LADC) is a major subtype of lung cancer, particularly among populations of East Asia. The epidermal growth factor receptor (EGFR) is the most frequently mutated oncogene promoting LADC progression and can serve as a therapeutic target in LADC. The tissue inhibitor of metalloproteinases (TIMP)-3 is a major regulator of extracellular matrix turnover via targeting of matrix metalloproteinases (MMPs), and thus, plays a critical role in tumor development and progression. The purpose of this study was to investigate potential associations among TIMP-3 genetic polymorphisms, EGFR statuses, and cancer clinicopathologic development in patients with LADC. In this study, 277 LADC patients with different EGFR statuses were recruited to dissect the allelic discrimination of TIMP-3 -1296 T>C (rs9619311), TIMP3 249T>C (rs9862), and TIMP3 261C>T (rs11547635) polymorphisms using a TaqMan allelic discrimination assay. Our data showed that compared to those LADC patients with wild-type CC homozygotes of TIMP-3 rs9862, patients harboring TT homozygotes of rs9862 were at a higher risk of developing mutant EGFR (adjusted odds ratio (AOR) = 2.530; 95% confidence interval (CI): 1.230–5.205; p = 0.012), particularly the EGFR L858R point mutation (AOR = 2.975; 95% CI: 1.182–7.488; p = 0.021). Moreover, we observed that TIMP-3 TT homozygotes of rs9862 were correlated with the incidence of EGFR mutations in patients with a smoking habit (p = 0.045). Within male patients harboring a mutant EGFR, TIMP-3 rs9862 T (CT+TT) allele carriers were at higher risk of developing an advanced stage (p = 0.025) and lymph node metastasis (p = 0.043). Further analyses of clinical datasets revealed correlations of TIMP-3 expression with a favorable prognosis in patients with LADC. In conclusion, the data suggest that TIMP-3 rs9862 polymorphisms may contribute to identify subgroups of lung cancer patients at high risk for tumor progression, among carriers of LADC-bearing mutant EGFR.

Curcumin (CUR) has a range of therapeutic benefits against cancers, but its poor solubility and low bioavailability limit its clinical use. Demethoxycurcumin (DMC) and diphenyl difluoroketone (EF-24) are natural and synthetic curcumin analogues, respectively, with better solubilities and higher anti-carcinogenic activities in various solid tumors than CUR. However, the efficacy of these analogues against non-solid tumors, particularly in acute myeloid leukemia (AML), has not been fully investigated. Herein, we observed that both DMC and EF-24 significantly decrease the proportion of viable AML cells including HL-60, U937, and MV4-11, harboring different NRAS and Fms-like tyrosine kinase 3 (FLT3) statuses, and that EF-24 has a lower half maximal inhibitory concentration (IC50) than DMC. We found that EF-24 treatment induces several features of apoptosis, including an increase in the sub-G1 population, phosphatidylserine (PS) externalization, and significant activation of extrinsic proapoptotic signaling such as caspase-8 and -3 activation. Mechanistically, p38 mitogen-activated protein kinase (MAPK) activation is critical for EF-24-triggered apoptosis via activating protein phosphatase 2A (PP2A) to attenuate extracellular-regulated protein kinase (ERK) activities in HL-60 AML cells. In the clinic, patients with AML expressing high level of PP2A have the most favorable prognoses compared to various solid tumors. Taken together, our results indicate that EF-24 is a potential therapeutic agent for treating AML, especially for cancer types that lose the function of the PP2A tumor suppressor.

Monoamine oxidases (MAOs) including MAOA and MAOB are enzymes located on the outer membranes of mitochondria, which are responsible for catalyzing monoamine oxidation. Recently, increased level of MAOs were shown in several cancer types. However, possible roles of MAOs have not yet been elucidated in the progression and prognosis of colorectal carcinoma (CRC). We therefore analyzed the importance of MAOs in CRC by an in silico analysis and tissue microarrays. Several independent cohorts indicated that high expression of MAOB, but not MAOA, was correlated with a worse disease stage and poorer survival. In total, 203 colorectal adenocarcinoma cases underwent immunohistochemical staining of MAOs, and associations with clinicopathological parameters and patient outcomes were evaluated. We found that MAOB is highly expressed in CRC tissues compared to normal colorectal tissues, and its expression was significantly correlated with a higher recurrence rate and a poor prognosis. Moreover, according to the univariate and multivariate analyses, we found that MAOB could be an independent prognostic factor for overall survival and disease-free survival, and its prognostic value was better than T and N stage. Furthermore, significant positive and negative correlations of MAOB with mesenchymal-type and epithelial-type gene expressions were observed in CRC tissues. According to the highlighted characteristics of MAOB in CRC, MAOB can be used as a novel indicator to predict the progression and prognosis of CRC patients.

Demethoxycurcumin (DMC) is a curcumin analogue with better stability and higher aqueous solubility than curcumin after oral ingestion and has the potential to treat diverse cancers, including oral squamous cell carcinoma (OSCC). The aim of this study was to investigate the anticancer effects and underlying mechanisms of DMC against OSCC. We found that DMC suppressed cell proliferation via simultaneously inducing G2/M-phase arrest and cell apoptosis. Mechanistic investigations found that the downregulation of cellular IAP 1 (cIAP1)/X-chromosome-linked IAP (XIAP) and upregulation of heme oxygenase-1 (HO-1) were critical for DMC-induced caspase-8/-9/-3 activation and apoptotic cell death. Moreover, p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK)1/2 were activated by DMC treatment in OSCC cells, and only the inhibition of p38 MAPK significantly abolished DMC-induced HO-1 expression and caspase-8/-9/-3 activation. The analyses of clinical datasets revealed that patients with head and neck cancers expressing high HO-1 and low cIAP1 had the most favorable prognoses. Furthermore, a combinatorial treatment of DMC with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, gefitinib, significantly enhanced the inhibitory effect of gefitinib on the proliferation of OSCC cells. Overall, the current study supported a role for DCM as part of a therapeutic approach for OSCC through suppressing IAPs and activating the p38-HO-1 axis.

[This corrects the article DOI: 10.1371/journal.pone.0031055.].

The long noncoding (lnc)RNA, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), plays a crucial role in the development of hepatocellular carcinoma (HCC). However, potential genetic variants (single nucleotide polymorphisms, SNPs) in MALAT1 that affect the susceptibility and progression of HCC have rarely been explored. Three tagging SNPs, viz., rs3200401 C > T, rs619586 A > G, and rs1194338 C > A, in MALAT1 were genotyped by a TaqMan allelic discrimination assay in 394 HCC patients and 1199 healthy controls. A stratified analysis showed that younger patients ( Furthermore, analyses of clinical datasets revealed that MALAT1 expression level was gradually unregulated during HCC development from normal liver, cirrhotic liver, dysplastic liver to HCC and correlated with poor survival rates in HCC patients, especially in the hepatitis virus-infected population.

Increasing evidence shows that dysregulated expression of long non-coding (lnc)RNAs can serve as diagnostic or prognostic markers in urothelial cell carcinoma (UCC), the most common pathological type of bladder cancer. lncRNA HOX transcript antisense RNA (HOTAIR) was shown to promote tumor progression and be associated with a poor prognosis in multiple cancers including bladder cancer. Polymorphisms of HOTAIR were recently linked to a predisposition for diverse malignancies. Herein we conducted a case-control study to evaluate whether genetic polymorphisms of HOTAIR were associated with UCC risk and clinicopathologic characteristics. Four loci (rs920778 T>C, rs1899663 G>T, rs4759314 A>G, and rs12427129, C>T) of HOTAIR were genotyped by a TaqMan allelic discrimination method in 431 cases and 862 controls. We found that female patients who carried AG + GG genotype of rs4759314 were associated with an increased UCC risk after controlling for age and tobacco consumption (adjusted odds ratio (AOR) = 1.92, 95% confidence interval (CI): 1.01⁻3.64, p = 0.047) and a lower overall survival rate (p = 0.008). Moreover, patients with a smoking habit or younger age (≤65 years), who had at least one T allele of HOTAIR rs12427129 were at a higher risk of developing advance tumor T satge (p = 0.046), compared to those patients with CC homozygotes. In contrast, rs920778 C allele carriers were negatively correlated with the development of lymph node metastasis (OR = 0.51, 95% CI: 0.28⁻0.94, p = 0.031). Further analyses of clinical datasets revealed correlations of the expression of HOTAIR with tumor metastasis and a poor survival rate in patients with UCC. Our results verified the diverse impacts of HOTAIR variants on UCC susceptibility and clinicopathologic characteristics.

Background: Glioblastoma (GBM), a malignant form of glioma, is characterized by resistance to therapy and poor prognosis. Accumulating evidence shows that the initiation, propagation, and recurrence of GBM is attributable to the presence of GBM stem cells (GBM-CSCs). Experimental approach: Herein, we investigated the effect of 4-Acetylantroquinonol B (4-AAQB), a bioactive isolate of Antrodia cinnamomea, on GBM cell viability, oncogenic, and CSCs-like activities. Results: We observed that aberrant expression of catenin is characteristic of GBM, compared to other glioma types (p = 0.0001, log-rank test = 475.2), and correlates with poor prognosis of GBM patients. Lower grade glioma and glioblastoma patients (n = 1152) with low catenin expression had 25% and 21.5% better overall survival than those with high catenin expression at the 5 and 10-year time-points, respectively (p = 3.57e-11, log-rank test = 43.8). Immunohistochemistry demonstrated that compared with adjacent non-tumor brain tissue, primary and recurrent GBM exhibited enhanced catenin expression (~10-fold, p < 0.001). Western blot analysis showed that 4-AAQB significantly downregulated β-catenin and dysregulated the catenin/LEF1/Stat3 signaling axis in U87MG and DBTRG-05MG cells, dose-dependently. 4-AAQB–induced downregulation of catenin positively correlated with reduced Sox2 and Oct4 nuclear expression in the cells. Furthermore, 4-AAQB markedly reduced the viability of U87MG and DBTRG-05MG cells with 48 h IC50 of 9.2 M and 12.5 M, respectively, effectively inhibited the nuclear catenin, limited the migration and invasion of GBM cells, with concurrent downregulation of catenin, vimentin, and slug; similarly, colony and tumorsphere formation was significantly attenuated with reduced expression of c-Myc and KLF4 proteins. Conclusions: Summarily, we show for the first time that 4-AAQB suppresses the tumor-promoting catenin/LEF1/Stat3 signaling, and inhibited CSCs-induced oncogenic activities in GBM in vitro, with in vivo validation; thus projecting 4-AAQB as a potent therapeutic agent for anti-GBM target therapy.

The purpose of this study was to explore the combined effect of melatonin receptor type 1A (MTNR1A) gene polymorphisms and exposure to environmental carcinogens on the susceptibility and clinicopathological characteristics of oral cancer.Three polymorphisms of the MTNR1A gene from 618 patients with oral cancer and 560 non-cancer controls were analyzed by real-time polymerase chain reaction (PCR). The CTA haplotype of the studied MTNR1A polymorphisms (rs2119882, rs13140012, rs6553010) was related to a higher risk of oral cancer. Moreover, MTNR1A gene polymorphisms exhibited synergistic effects of environmental factors (betel quid and tobacco use) on the susceptibility of oral cancer. Finally, oral-cancer patients with betel quid-chewing habit who had T/T allele of MTNR1A rs13140012 were at higher risk for developing an advanced clinical stage and lymph node metastasis.These results support gene-environment interactions of MTNR1A polymorphisms with smoking and betel quid-chewing habits possibly altering oral-cancer susceptibility and metastasis.