Xue Wen

A novel antisense long noncoding RNA within the IGF1R gene locus is imprinted in hematopoietic malignancies

Dysregulation of the insulin-like growth factor type I receptor (IGF1R) has been implicated in the progression and therapeutic resistance of malignancies. In acute myeloid leukemia (AML) cells, IGF1R is one of the most abundantly phosphorylated receptor tyrosine kinases, promoting cell growth through the PI3K/Akt signaling pathway. However, little is known regarding the molecular mechanisms underlying IGF1R gene dysregulation in cancer. We discovered a novel intragenic long noncoding RNA (lncRNA) within the IGF1R locus, named IRAIN, which is transcribed in an antisense direction from an intronic promoter. The IRAIN lncRNA was expressed exclusively from the paternal allele, with the maternal counterpart being silenced. Using both reverse transcription-associated trap and chromatin conformation capture assays, we demonstrate that this lncRNA interacts with chromatin DNA and is involved in the formation of an intrachromosomal enhancer/promoter loop. Knockdown of IRAIN lncRNA with shRNA abolishes this intrachromosomal interaction. In addition, IRAIN was downregulated both in leukemia cell lines and in blood obtained from high-risk AML patients. These data identify IRAIN as a new imprinted lncRNA that is involved in long-range DNA interactions.

Loss of insulin-like growth factor II imprinting is a hallmark associated with enhanced chemo/radiotherapy resistance in cancer stem cells

Insulin-like growth factor II (IGF2) is maternally imprinted in most tissues, but the epigenetic regulation of the gene in cancer stem cells (CSCs) has not been defined. To study the epigenetic mechanisms underlying self-renewal, we isolated CSCs and non-CSCs from colon cancer (HT29, HRT18, HCT116), hepatoma (Hep3B), breast cancer (MCF7) and prostate cancer (ASPC) cell lines. In HT29 and HRT18 cells that show loss of IGF2 imprinting (LOI), IGF2 was biallelically expressed in the isolated CSCs. Surprisingly, we also found loss of IGF2 imprinting in CSCs derived from cell lines HCT116 and ASPC that overall demonstrate maintenance of IGF2 imprinting. Using chromatin conformation capture (3C), we found that intrachromosomal looping between the IGF2 promoters and the imprinting control region (ICR) was abrogated in CSCs, in parallel with loss of IGF2 imprinting in these CSCs. Loss of imprinting led to increased IGF2 expression in CSCs, which have a higher rate of colony formation and greater resistance to chemotherapy and radiotherapy in vitro. These studies demonstrate that IGF2 LOI is a common feature in CSCs, even when the stem cells are derived from a cell line in which the general population of cells maintain IGF2 imprinting. This finding suggests that aberrant IGF2 imprinting may be an intrinsic epigenetic control mechanism that enhances stemness, self-renewal and chemo/radiotherapy resistance in cancer stem cells.

Mitochondrial DNA Hypomethylation Is a Biomarker Associated with Induced Senescence in Human Fetal Heart Mesenchymal Stem Cells

Background. Fetal heart can regenerate to restore its normal anatomy and function in response to injury, but this regenerative capacity is lost within the first week of postnatal life. Although the specific molecular mechanisms remain to be defined, it is presumed that aging of cardiac stem or progenitor cells may contribute to the loss of regenerative potential. Methods. To study this aging-related dysfunction, we cultured mesenchymal stem cells (MSCs) from human fetal heart tissues. Senescence was induced by exposing cells to chronic oxidative stress/low serum. Mitochondrial DNA methylation was examined during the period of senescence. Results. Senescent MSCs exhibited flattened and enlarged morphology and were positive for the senescence-associated beta-galactosidase (SA-β-Gal). By scanning the entire mitochondrial genome, we found that four CpG islands were hypomethylated in close association with senescence in MSCs. The mitochondrial COX1 gene, which encodes the main subunit of the cytochrome c oxidase complex and contains the differentially methylated CpG island 4, was upregulated in MSCs in parallel with the onset of senescence. Knockdown of DNA methyltransferases (DNMT1, DNMT3a, and DNMT3B) also upregulated COX1 expression and induced cellular senescence in MSCs. Conclusions. This study demonstrates that mitochondrial CpG hypomethylation may serve as a critical biomarker associated with cellular senescence induced by chronic oxidative stress.

Friend leukemia virus integration 1 promotes tumorigenesis of small cell lung cancer cells by activating the miR-17-92 pathway

Small cell lung cancer (SCLC) is regarded as the most devastative type of human lung malignancies. The rapid and disseminated growth pattern remains the primary cause of poor clinical prognosis in patients with SCLC. However, the molecular factors that drive rapid progression of SCLC remain unclear. Friend leukemia virus integration 1 (FLI1), an Ets transcription factor family member, has been previously reported to act as a major driver of hematological malignancies. In this study, we explored the potential role of FLI1 in SCLC. Using immunohistochemical staining, we found that FLI1 was significantly upregulated in SCLC tissues, compared to that in non-small cell lung cancer (NSCLC) and normal lung tissues (p < 0.01). The expression score of FLI1 oncoprotein was associated with the extensive stage of SCLC and the overexpressed Ki67. Knockdown of FLI1 with small interfering RNA (siRNA) or short hairpin RNA (shRNA) promoted apoptosis and induced repression of cell proliferation, tumor colony formation and in vivo tumorigenicity in highly aggressive SCLC cell lines. Importantly, we discovered that FLI1 promoted tumorigenesis by activating the miR-17-92 cluster family. This study uncovers FLI1 as an important driving factor that promotes tumor growth in SCLC through the miR-17-92 pathway. FLI1 may serve as an attractive target for therapeutic intervention of SCLC.

Distinct biological effects of low-dose radiation on normal and cancerous human lung cells are mediated by ATM signaling

Low-dose radiation (LDR) induces hormesis and adaptive response in normal cells but not in cancer cells, suggesting its potential protection of normal tissue against damage induced by conventional radiotherapy. However, the underlying mechanisms are not well established. We addressed this in the present study by examining the role of the ataxia telangiectasia mutated (ATM) signaling pathway in response to LDR using A549 human lung adenocarcinoma cells and HBE135-E6E7 (HBE) normal lung epithelial cells. We found that LDR-activated ATM was the initiating event in hormesis and adaptive response to LDR in HBE cells. ATM activation increased the expression of CDK4/CDK6/cyclin D1 by activating the AKT/glycogen synthase kinase (GSK)-3β signaling pathway, which stimulated HBE cell proliferation. Activation of ATM/AKT/GSK-3β signaling also increased nuclear accumulation of nuclear factor erythroid 2-related factor 2, leading to increased expression of antioxidants, which mitigated cellular damage from excessive reactive oxygen species production induced by high-dose radiation. However, these effects were not observed in A549 cells. Thus, the failure to activate these pathways in A549 cells likely explains the difference between normal and cancer cells in terms of hormesis and adaptive response to LDR.

Validating the pivotal role of the immune system in low-dose radiation-induced tumor inhibition in Lewis lung cancer-bearing mice

Although low‐dose radiation (LDR) possesses the two distinct functions of inducing hormesis and adaptive responses, which result in immune enhancement and tumor inhibition, its clinical applications have not yet been elucidated. The major obstacle that hinders the application of LDR in the clinical setting is that the mechanisms underlying induction of tumor inhibition are unclear, and the risks associated with LDR are still unknown. Thus, to overcome this obstacle and elucidate the mechanisms mediating the antitumor effects of LDR, in this study, we established an in vivo lung cancer model to investigate the participation of the immune system in LDR‐induced tumor inhibition and validated the pivotal role of the immune system by impairing immunity with high‐dose radiation (HDR) of 1 Gy. Additionally, the LDR‐induced adaptive response of the immune system was also observed by sequential HDR treatment in this mouse model. We found that LDR‐activated T cells and natural killer cells and increased the cytotoxicity of splenocytes and the infiltration of T cells in the tumor tissues. In contrast, when immune function was impaired by HDR pretreatment, LDR could not induce tumor inhibition. However, when LDR was administered before HDR, the immunity could be protected from impairment, and tumor growth could be inhibited to some extent, indicating the induction of the immune adaptive response by LDR. Therefore, we demonstrated that immune enhancement played a key role in LDR‐induced tumor inhibition. These findings emphasized the importance of the immune response in tumor radiotherapy and may help promote the application of LDR as a novel approach in clinical practice.

Bioinformatics analyses of differentially expressed genes associated with bisphosphonate-related osteonecrosis of the jaw in patients with multiple myeloma.

Purpose This study aimed to explore the molecular mechanisms associated with bisphosphonate (BP)-related osteonecrosis of the jaw (ONJ) in patients with multiple myeloma (MM). Methods The gene expression profile GSE7116 was downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) from eleven patients with ONJ resulting from MM treated with BPs (ONJBPs) and ten MM patients without ONJ treated with BPs (MMBPs) were analyzed. Gene ontology (GO) and pathway enrichment analyses of DEGs were performed, followed by functional annotation and protein–protein interaction network construction. Finally, sub-network modules were constructed and analyzed. Results A total of 166 up- and 473 down-regulated DEGs were identified. The up-regulated DEGs were enriched in pathways related to cancer, and the down-regulated DEGs were enriched in pathways related to the immune system. Moreover, the GO terms enriched by the up-regulated DEGs were associated with misfolded proteins, and the down-regulated DEGs were associated with immune responses. After functional annotation, 16 transcription factors were identified, including X-box binding protein 1 (XBP1). In protein–protein interaction network analysis, tumor necrosis factor (TNF) and interleukin 1, beta (IL1B) had higher connectivity degrees. Among the constructed sub-network modules, module 1 was the best one, and DEAD (Asp-Glu-Ala-Asp) box helicase 5 (DDX5) was a hub gene. The DEGs in module 1 were mainly enriched in GO terms related to RNA splicing. Conclusion DEGs of ONJ were mainly enriched in pathways related to the immune system and RNA splicing. DEGs such as TNF, ILB1, DDX5, and XBP1 may be the potential targets of ONJ treatment.

Targeted breast cancer therapy by harnessing the inherent blood group antigen immune system

Cancer gene therapy has attracted increasing attention for its advantages over conventional therapy in specific killing of tumor cells. Here, we attempt to prove a novel therapeutic approach that targets tumors by harnessing the blood antigen immune response system, which is inherently present in patients with breast cancers. Breast cancer MDA-MB-231 cells expressed blood group H antigen precursor. After ectopic expression of blood group A glycosyltransferase, we found that the H precursor was converted into the group A antigen, appearing on the surface of tumor cells. Incubation with group B plasma from breast cancer patients activated the antigen-antibody-complement cascade and triggered tumor cell killing. Interestingly, expression of blood A antigen also reduced tumorigenesis in breast cancer cells by inhibiting cell proliferation, migration, and tumor sphere formation. Cell cycle analysis revealed that cancer cells were paused at S phase due to the activation of cell cycle regulatory genes. Furthermore, pro-apoptotic genes were unregulated by the A antigen, including BAX, P21, and P53, while the anti-apoptotic BCL2 was down regulated. Importantly, we showed that extracellular HMGB1 and ATP, two critical components of the immunogenic cell death pathway, were significantly increased in the blood A antigen-expressing tumor cells. Collectively, these data suggest that blood antigen therapy induces specific cancer cell killing by activating the apoptosis and immunogenic cell death pathways. Further translational studies are thereby warranted to apply this approach in cancer immuno-gene therapy.

Targeting the IGF1R Pathway in Breast Cancer Using Antisense lncRNA-Mediated Promoter cis Competition

Aberrant insulin-like growth factor I receptor (IGF1R) signaling pathway serves as a well-established target for cancer drug therapy. The intragenic antisense long noncoding RNA (lncRNA) IRAIN, a putative tumor suppressor, is downregulated in breast cancer cells, while IGF1R is overexpressed, leading to an abnormal IGF1R/IRAIN ratio that promotes tumor growth. To precisely target this pathway, we developed an “antisense lncRNA-mediated intragenic cis competition” (ALIC) approach to therapeutically correct the elevated IGF1R/IRAIN bias in breast cancer cells. We used CRISPR-Cas9 gene editing to target the weak promoter of IRAIN antisense lncRNA and showed that in targeted clones, intragenic activation of the antisense lncRNA potently competed in cis with the promoter of the IGF1R sense mRNA. Notably, the normalization of IGF1R/IRAIN transcription inhibited the IGF1R signaling pathway in breast cancer cells, decreasing cell proliferation, tumor sphere formation, migration, and invasion. Using “nuclear RNA reverse transcription-associated trap” sequencing, we uncovered an IRAIN lncRNA-specific interactome containing gene targets involved in cell metastasis, signaling pathways, and cell immortalization. These data suggest that aberrantly upregulated IGF1R in breast cancer cells can be precisely targeted by cis transcription competition, thus providing a useful strategy to target disease genes in the development of novel precision medicine therapies.

A Novel Inherited Mutation in PRKAR1A Abrogates PreRNA Splicing in a Carney Complex Family

BACKGROUND Carney complex (CNC) is an autosomal dominant inherited disease, characterized by spotty skin pigmentation, cardiac and cutaneous myxomas, and endocrine overactivity. We report on a Chinese CNC family with a novel mutation in the protein kinase A regulatory subunit 1 (PRKAR1A) gene. METHODS Target-exome sequencing was performed to identify the mutation of PRKAR1A in 2 members of the CNC family. RESULTS The proband was a young man with typical CNC, including pigmentation, cutaneous myxomas, cardiac myxoma, Sertoli cell tumour of his left testis, and multiple hypoechoic thyroid nodules. His mother also had CNC with skin pigmentation, cutaneous myxomas, and a cardiac myxoma. Target-exome capture analysis revealed that the proband and the mother carried a novel heterozygous mutation in the exon 6 splicing donor site of PRKAR1A. Sequencing analysis of myxoma messenger RNA revealed that the mutation abrogated exon 6 preRNA splicing, leading to a frameshift starting at Valine 185 and premature translation termination in intron 6. The truncated enzyme lacks the functional cyclic adenosine monophosphate (cAMP) binding domain at the C-terminus, causing PRKAR1A haploinsufficiency. CONCLUSIONS In this study we report on a novel splicing mutation in the PRKAR1A gene that adds to the genetic heterogeneity of CNC.