Selective Inhibition of PI3K Isoforms in Brain Tumors Suppresses Tumor Growth by Increasing Radiosensitivity.

PURPOSE: Glioblastoma (GBM) is a malignant brain tumor with poor prognosis. Radioresistance is a major challenge in the treatment of brain tumors. The development of several types of tumors, including GBM, involves the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. Upon activation, this pathway induces radioresistance. In this study, we investigated whether additional use of selective inhibitors of PI3K isoforms would enhance radiosensitivity in GBM. MATERIALS AND METHODS: We evaluated whether radiation combined with PI3K isoform selective inhibitors can suppress radioresistance in GBM. Glioma 261 expressing luciferase (GL261-luc) and LN229 were used to confirm the effect of combination of radiation and PI3K isoform inhibitors in vitro. Cell viability was confirmed by clonogenic assay, and inhibition of PI3K/AKT signaling activation was observed by Western blot. To confirm radiosensitivity, the expression of phospho-γ-H2AX was observed by immunofluorescence. In addition, to identify the effect of a combination of radiation and PI3K-α isoform inhibitor in vivo, an intracranial mouse model was established by implanting GL261-luc. Tumor growth was observed by IVIS imaging, and survival was analyzed using Kaplan-Meier survival curves. RESULTS: Suppression of the PI3K/AKT signaling pathway increased radiosensitivity, and PI3K-α inhibition had similar effects on PI3K-pan inhibition in vitro. The combination of radiotherapy and PI3K-α isoform inhibitor suppressed tumor growth and extended survival in vivo. CONCLUSION: This study verified that PI3K-α isoform inhibition improves radiosensitivity, resulting in tumor growth suppression and extended survival in GBM mice.

Combining radiation with PI3K isoform-selective inhibitor administration increases radiosensitivity and suppresses tumor growth in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is a malignant lung tumor with a dismal prognosis. The activation of the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway is common in many tumor types including NSCLC, which results in radioresistance and changes in the tumor microenvironment. Although pan-PI3K inhibitors have been tested in clinical trials to overcome radioresistance, concerns regarding their excessive side effects led to the consideration of selective inhibition of PI3K isoforms. In this study, we assessed whether combining radiation with the administration of the PI3K isoform-selective inhibitors reduces radioresistance and tumor growth in NSCLC. Inhibition of the PI3K/AKT pathway enhanced radiosensitivity substantially, and PI3K-α inhibitor showed superior radiosensitizing effect similar to PI3K pan-inhibitor, both in vitro and in vivo. Additionally, a significant increase in DNA double-strand breaks (DSB) and a decrease in migration ability were observed. Our study revealed that combining radiation and the PI3K-α isoform improved radiosensitivity that resulted in a significant delay in tumor growth and improved survival rate.

Coupling of LETM1 up-regulation with oxidative phosphorylation and platelet-derived growth factor receptor signaling via YAP1 transactivation.

Persistent cellular proliferation and metabolic reprogramming are essential processes in carcinogenesis. Here, we performed Gene Set Enrichment Analysis (GSEA) and found that that LETM1, a mitochondrial calcium transporter, is associated with cellular growth signals such as platelet-derived growth factor (PDGF) receptor signaling and insulin signaling pathways. These results were then verified by qRT-PCR and immnunoblotting. Mechanistically, up-regulation of LETM1 induced YAP1 nuclear accumulation, increasing the expression of PDGFB, PDGFRB and THBS4. Consistent with this, LETM1 silencing caused loss of YAP1 nuclear signal, decreasing the expression of PDGFB, PDGFRB and THBS4. Immunohistochemical staining consistently indicated a positive association between LETM1 up-regulation, YAP1 nuclear localization and high PDGFB expression. In clinical data analysis, LETM1 up-regulation in thyroid cancer was found to be related to aggressive tumor features such as lymphovascular invasion (LVI, P < 0.001) and lymph node metastasis (LNM, P = 0.011). Multivariate analysis demonstrated that LETM1 up-regulation increases the risk of LVI and LNM (OR = 3.455, 95% CI = 1.537-7.766 and OR = 3.043, 95% CI = 1.282-7.225, respectively). Collectively, these data suggest that up-regulation of LETM1 induces sustained activation of proliferative signaling pathways, such as PDGF signal pathway by AKT induced YAP1 transactivation, resulting in aggressive thyroid cancer phenotypes.

Upregulation of long noncoding RNA LOC100507661 promotes tumor aggressiveness in thyroid cancer.

Recent advances in next-generation sequencing have revealed a variety of long noncoding RNAs (lncRNAs). However, studies of lncRNAs are at a very early stage, our knowledge of the biological functions and clinical implications remains limited. To investigate the roles of lncRNAs in thyroid cancers, we verified 56 lncRNAs identified as potential cancer-promoting genes in a previous study that analyzed 2394 tumor SNP arrays from 12 types of cancer. Based on verified sequence information in NCBI and Ensembl, we ultimately selected three candidate lncRNAs for detailed analysis. One of the candidates, LOC100507661, was strongly upregulated in thyroid cancer tissues relative to paired contralateral normal tissue. LOC100507661 was easily detectable in papillary and anaplastic thyroid cancer cell lines such as TPC1, BCPAP, C643, and 8505C, but not in the follicular thyroid cancer cell line FTC133. Stable overexpression of LOC100507661 promoted cell proliferation, migration, and invasion of thyroid cancer cells. Lymph node metastasis and BRAF V600E mutations were more frequent in papillary thyroid cancers with high LOC100507661 expression. Our data demonstrate that LOC100507661 expression is elevated in human thyroid cancer and may play a critical role in thyroid carcinogenesis.

Distinct Features of Nonthyroidal Illness in Critically Ill Patients With Infectious Diseases.

Nonthyroidal illness (NTI), often observed in critically ill patients, arises through diverse alterations in the hypothalamus-pituitary-thyroid (HPT) axis. However, the causal relationship between underlying disease and NTI diversity in critically ill patients is poorly understood.The aim of this study was to examine NTI severity and adverse outcomes in critically ill patients with respect to their underlying disease(s).The medical records of 616 patients admitted to the intensive care unit (ICU) between January 2009 and October 2014 were retrospectively reviewed. Patients with known diseases or taking medications that affect thyroid function were excluded. All-cause mortality (ACM) and length of stay (LOS) in the ICU were assessed as adverse outcomes.The enrolled patients (n = 213) were divided into the following 4 groups according to the severity of NTI at the nadir of their thyroid function test (TFT): normal (n = 11, 5.2%), mild NTI (n = 113, 53.1%), moderate NTI (n = 78, 36.6%), and severe NTI (n = 11, 5.2%). There was no significant difference between the groups in terms of age and gender. NTI severity showed a significantly strong association with ACM (P < 0.0001) and a significant positive association with LOS in the ICU (P = 0.031). After adjusting for age, gender, and current medications affecting TFT, increasing NTI severity led to increased ACM (odds ratio = 3.101; 95% confidence interval = 1.711-5.618; P < 0.0001). Notably, the prevalence of moderate-to-severe NTI was markedly higher in patients with infectious disease than in those with noninfectious disease (P = 0.012). Consistent with this, serum C-reactive protein levels were higher in patients with moderate-to-severe NTI (P = 0.016).NTI severity is associated with increased ACM, LOS, and underlying infectious disease. Future studies will focus on the biological and clinical implications of infectious disease on the HPT axis.

Relationship of Focally Amplified Long Noncoding on Chromosome 1 (FAL1) lncRNA with E2F Transcription Factors in Thyroid Cancer.

Recent functional genomic studies revealed that the oncogenic activity of focally amplified lncRNA on chromosome 1 (FAL1, ENSG00000228126) contributes to tumor growth by p21 repression in human cancers. However, the expression of FAL1 was not investigated in papillary thyroid cancer (PTC). We aimed to determine if FAL1 was up-regulated in PTC compared to paired contralateral normal thyroid tissues, and to investigate the potential targets of this lncRNA and its clinicopathological significance in PTC. We analyzed FAL1 and p21 expression levels in 100 PTC samples and matched normal thyroid tissue by qRT-PCR. Using lncRNA microarray data from the Gene Expression Omnibus (accession no. GSE61763), we explored potential targets of FAL1 by Gene Set Enrichment Analysis, followed by verification by qRT-PCR in our PTC samples. A cross-sectional observational study was conducted to investigate the relationship between patients' clinicopathological features and FAL1 expression. FAL1 expression was significantly higher in PTC than in paired normal thyroid tissues (paired t test, P < 0.001). p21 mRNA expression was also increased, not decreased, in PTC, and had no correlation with FAL1 expression (r = 0.0897, P = 0.4002). Gene Set Enrichment Analysis, using publicly available microarray data, indicated that a gene set related to the cell cycle, including E2F transcription factors 1 and 2, and cyclin D1, was coordinately enriched among samples with high FAL1 expression. A volcano plot showed that E2F1, E2F2, and VEGFA mRNAs were increased in the high FAL1 samples. In clinicopathological analyses, multifocality was more frequently observed in PTC patients with high FAL1 (P = 0.018). Multivariate analysis showed that high FAL1 expression increased the risk of multifocality (after adjustment for clinical variables, OR = 4.019, CI = 1.041-11.020, P = 0.043). FAL1 may have a role in cell-cycle progression and may be associated with aggressive tumor behavior in PTC.

KSR1 is coordinately regulated with Notch signaling and oxidative phosphorylation in thyroid cancer.

Kinase suppressor of RAS1 (KSR1) is a scaffold protein implicated in RAS-mediated RAF activation. However, the molecular function of KSR in papillary thyroid cancer (PTC) is unknown. Thus, this study aimed to characterize the role of KSR1 in patients with PTC. qRT-PCR and immunohistochemistry (IHC) revealed inter-tumor heterogeneities in the expression of KSR1 in PTC tissues. Interestingly, BRAFV600E-positive PTC showed higher KSR1 mRNA expression than BRAFV600E-negative PTC (P<0.001). Gene Set Enrichment Analysis (GSEA) using public repositories showed that high KSR1 expression coordinately upregulated Notch signaling (nominal P=0.019, false discovery rate (FDR) q-value=0.165); this finding was supported by GeneNetwork analysis, indicating that KSR1 expression is positively correlated with NOTCH1 expression (ρ=0.677, P=6.15×10(-9)). siRNA against KSR1 (siKSR1) significantly decreased ERK phosphorylation induced by BRAFV600E, resulting in reduced expression of NOTCH1 and HES1, targets of Notch signaling. GSEA revealed that high KSR1 expression was also associated with downregulation of genes related to oxidative phosphorylation (OxPhos). Consistent with this, electron microscopy showed that PTCs with high KSR1 expression exhibited structural defects of the mitochondrial cristae. Furthermore, siKSR1-transfected BCPAP and 8505C cells generated fewer colonies in colony-forming assays. In addition, GSEA showed that high expression of KSR2 and connector enhancer of KSR1 (CNKSR1) also coordinately upregulated Notch signaling (KSR2: nominal P=0.0097, FDR q-value=0.154 and CNKSR1: nominal P<0.0001, FDR q-value=0.00554), and high CNKSR2 was associated with downregulation of the OxPhos gene set (nominal P<0.0001, FDR q-value <0.0001). In conclusion, KSR1 is coordinately regulated with Notch signaling and OxPhos in PTC, because its scaffold function might be required to sustain the proliferative signaling and metabolic remodeling associated with this type of cancer.

A metabolic phenotype based on mitochondrial ribosomal protein expression as a predictor of lymph node metastasis in papillary thyroid carcinoma.

Metabolic reprogramming has been regarded as an essential component of malignant transformation. However, the clinical significance of metabolic heterogeneity remains poorly characterized. The aim of this study was to characterize metabolic heterogeneity in thyroid cancers via the analysis of the expression of mitochondrial ribosomal proteins (MRPs) and genes involved in oxidative phosphorylation (OxPhos), and investigate potential prognostic correlations. Gene set enrichment analysis (GSEA) verified by reverse transcription polymerase chain reaction and gene network analysis was performed using public repository data. Cross-sectional observational study was conducted to classify papillary thyroid cancer (PTC) by the expression of MRP L44 (MRPL44) messenger RNA (mRNA), and to investigate the clinicopathological features. GSEA clearly showed that the expression of OxPhos and MRP gene sets was significantly lower in primary thyroid cancer than in matched normal thyroid tissue. However, 8 of 49 primary thyroid tumors (16.3%) in the public repository did not show a reduction in OxPhos mRNA expression. Remarkably, strong positive correlations between MRPL44 expression and those of OxPhos and MRPs such as reduced nicotinamide adenine dinucleotide dehydrogenase (ubiquinone) 1 α subcomplex, 5; succinate dehydrogenase complex, subunit D; cytochrome c, somatic; adenosine triphosphate synthase, H+ transporting, mitochondrial Fo complex, subunit C1 (subunit 9); and MRP S5 (MRPS5) (P < 0.0001) were clearly denoted, suggesting that MRPL44 is a representative marker of OxPhos and MRP expressions. In laboratory experiments, metabolic heterogeneity in oxygen consumption, extracellular acidification rates (ECARs), and amounts of OxPhos complexes were consistently observed in BCPAP, TPC1, HTH-7, and XTC.UC1 cell lines. In PTCs, metabolic phenotype according to OxPhos amount defined by expression of MRPL44 mRNA was significantly related to lymph node metastasis (LNM) (P < 0.001). Furthermore, multivariate analysis clearly indicated that expression of MRPL44 is associated with an increased risk of lateral neck LNM (odds ratio 9.267, 95% confidence interval 1.852-46.371, P = 0.007). MRPL44 expression may be a representative marker of metabolic phenotype according to OxPhos amount and a useful predictor of LNM.

Bobby Sox homology regulates odontoblast differentiation of human dental pulp stem cells/progenitors.

BACKGROUND: Transcription factors have been implicated in regulating the differentiation of odontoblasts from dental pulp stem cells/progenitors (DPSCs/progenitors), but their regulatory network is not completely understood. RESULT: New transcription factors that control the odontoblast differentiation of human DPSCs/progenitors were analyzed using a microarray. The result revealed bobby sox homolog (BBX) to be expressed most strongly during odontoblast differentiation. Validation using RT-PCR also revealed the strong expression of BBX during the odontoblast differentiation of DPSCs/progenitors. BBX expression was also detected in adult molar odontoblasts and other tissues, including the heart, kidney, testis, and bone marrow. To understand the role of BBX in odontoblast differentiation, BBX variant 1 and 2 cDNA were cloned and overexpressed in DPSCs/progenitors. The results showed that the overexpression of BBX cDNA in DPSCs/progenitors induced substantial mineralization and expression of the odontoblast marker genes, such as ALP, OPN, BSP, DMP1, and DSPP. The knockdown of BBX using shRNA, however, did not affect mineralization, but the expression of ALP and DSPP was decreased substantially. Meanwhile overexpression or knockdown of BBX did not modulate proliferation of DPSCs/progenitors. CONCLUSION: Our results suggest that BBX plays an important role during the odontoblast differentiation of human DPSCs/progenitors.