tRF-Leu reverse breast cancer cells chemoresistance by regulation of BIRC5.

OBJECTIVE: Accumulating studies reported the crucial roles of tRFs in tumorigenesis. However, their further mechanisms and clinical values remains unclear. This study aimed at the further investigation of tRF-Leu in breast cancer chemotherapy resistance. METHODS: The high-throughput sequencing was performed and identified the downregulation of tRF-Leu in MCF7/ADR cells. The function of tRF-Leu in breast cancer cells and breast cancer chemotherapy resistance was investigated in vitro and in vivo, including colony formation assay, CCK-8 assay, transwell assay and apoptosis assay. The binding site of tRF-Leu on BIRC5 was verified by dual-luciferase assay. RESULTS: tRF-Leu was downregulated in MCF7/ADR cells. Overexpression of tRF-Leu inhibited the migration of breast cancer cells. Furthermore, tRF-Leu could reverse the resistance of MCF7/ADR cells to Adriamycin both in vitro and in vivo. BIRC5 was a target of tRF-Leu, which might be involved in the chemotherapy resistance regulation. CONCLUSION: We demonstrated that tRF-Leu could inhibit the chemotherapy resistance of breast cancer by targeting BIRC5. These findings might identify new biomarkers of breast cancer therapy and bring new strategies to reverse chemotherapy resistance.

Decreased BIRC5-206 promotes epithelial-mesenchymal transition in nasopharyngeal carcinoma through sponging miR-145-5p.

Metastasis significantly contributes to the poor prognosis of advanced nasopharyngeal carcinoma (NPC). Our prior studies have demonstrated a decrease in BIRC5-206 expression in NPC, which promotes disease progression. However, the role of BIRC5-206 in the invasion and metastasis of NPC has not been fully elucidated. In this study, our objective was to explore the biological function and underlying mechanisms of BIRC5-206 in NPC. Additionally, we established an NPC mouse model of lung invasiveness using C666 cells to assess the impact of BIRC5-206 on NPC metastasis. Our results revealed that silencing BIRC5-206 inhibited apoptosis and enhanced the invasion of NPC cells, whereas its overexpression reversed these effects. Moreover, decreased BIRC5-206 expression significantly increased N-cadherin and Vimentin expression while reducing E-cadherin and occludin levels, both in vivo and in vitro. Additionally, silencing BIRC5-206 markedly augmented the formation of invasive foci in lung tissues. Rescue experiments further confirmed that decreased BIRC5-206 expression facilitates NPC metastasis via modulation of the miR-145-5p/CD40 signaling pathway. In summary, our study suggests that BIRC5-206 may serve as a potential prognostic biomarker and therapeutic target in the diagnosis and treatment of NPC.

Robust aptamer-targeted CRISPR/Cas9 delivery using mesenchymal stem cell membrane -liposome hybrid: BIRC5 gene knockout against melanoma.

In this study, a platform was fabricated by combining a cationic lipid, 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) with mesenchymal stem cell membrane (MSCM) to produce a positively charged hybrid vesicle. The prepared hybrid vesicle was used to condense BIRC5 CRISPR/Cas9 plasmid for survivin (BIRC5) gene editing. The Sgc8-c aptamer (against protein tyrosine kinase 7) was then attached to the surface of the prepared NPs through electrostatic interactions. In this regard, melanoma cancer cells (B16F0 cell line) overexpressing PTK7 receptor could be targeted. Investigations were conducted on this system to evaluate its transfection efficiency, cellular toxicity, and therapeutic performance in preclinical stage using B16F0 tumor bearing C57BL/6 J mice. The results verified the superiority of the Hybrid/ BIRC5 compared to Liposome/ BIRC5 in terms of cellular toxicity and transfection efficiency. The cells exposure to Hybrid/BIRC5 significantly enhanced cytotoxicity. Moreover, cells treated with Apt-Hybrid/BIRC5 showed higher anti-proliferation activity toward PTK7-positive B16F0 cancer cells than that of the PKT7-negative CHO cell line. The active tumor targeting nanoparticles increased the cytotoxicity through down-regulation of BIRC5 expression as confirmed by Western blot analysis. In preclinical stage, Apt-Hybrid/BIRC5 showed remarkable tumor growth suppression toward B16F0 tumorized mice. Thus, our study suggested that genome editing for BIRC5 through the CRISPR/Cas9 system could provide a potentially safe approach for melanoma cancer therapy and has great potential for clinical translation.

Anti-BIRC5 autoantibody serves as a valuable biomarker for diagnosing AFP-negative hepatocellular carcinoma.

Background: Autoantibodies targeting tumor-associated antigens (TAAbs) have emerged as promising biomarkers for early cancer detection. This research aimed to assess the diagnostic capacity of anti-BIRC5 autoantibody in detecting AFP-negative hepatocellular carcinoma (ANHCC). Methods: This research was carried out in three stages (discovery phase, validation phase, and evaluation phase) and included a total of 744 participants. Firstly, the anti-BIRC5 autoantibody was discovered using protein microarray, exhibiting a higher positive rate in ANHCC samples (ANHCCs) compared to normal control samples (NCs). Secondly, the anti-BIRC5 autoantibody was validated through enzyme-linked immunosorbent assay (ELISA) in 85 ANHCCs and 85 NCs from two clinical centers (Zhengzhou and Nanchang). Lastly, the diagnostic usefulness of the anti-BIRC5 autoantibody for hepatocellular carcinoma (HCC) was evaluated by ELISA in a cohort consisting of an additional 149 AFP-positive hepatocellular carcinoma samples (APHCCs), 95 ANHCCs and 244 NCs. The association of elevated autoantibody to high expression of BIRC5 in HCC was further explored by the database from prognosis, immune infiltration, DNA methylation, and gene mutation level. Results: In the validation phase, the area under the ROC curve (AUC) of anti-BIRC5 autoantibody to distinguish ANHCCs from NCs in Zhengzhou and Nanchang centers was 0.733 and 0.745, respectively. In the evaluation phase, the AUCs of anti-BIRC5 autoantibody for identifying ANHCCs and HCCs from NCs were 0.738 and 0.726, respectively. Furthermore, when combined with AFP, the AUC for identifying HCCs from NCs increased to 0.914 with a sensitivity of 77.5% and specificity of 91.8%. High expression of BIRC5 gene is not only correlated with poor prognosis of HCCs, but also significantly associated with infiltration of immune cells, DNA methylation, and gene mutation. Conclusion: The findings suggest that the anti-BIRC5 autoantibody could serve as a potential biomarker for ANHCC, in addition to its supplementary role alongside AFP in the diagnosis of HCC. Next, we can carry out specific verification and explore the function of anti-BIRC5 autoantibody in the occurrence and development of HCC.

Targeting BIRC5 as a therapeutic approach to overcome ASXL1-associated decitabine resistance.

Hypomethylating agents (HMAs) are widely employed in the treatment of myeloid malignancies. However, unresponsive or resistant to HMAs occurs in approximately 50 % of patients. ASXL1, one of the most commonly mutated genes across the full spectrum of myeloid malignancies, has been reported to predict a lower overall response rate to HMAs, suggesting an essential need to develop effective therapeutic strategies for the patients with HMA failure. Here, we investigated the impact of ASXL1 on cellular responsiveness to decitabine treatment. ASXL1 deficiency increased resistance to decitabine treatment in AML cell lines and mouse bone marrow cells. Transcriptome sequencing revealed significant alterations in genes regulating cell cycle, apoptosis, and histone modification in ASXL1 deficient cells that resistant to decitabine. BIRC5 was identified as a potential target for overcoming decitabine resistance in ASXL1 deficient cells. Furthermore, our experimental evidence demonstrated that the small-molecule inhibitor of BIRC5 (YM-155) synergistically sensitized ASXL1 deficient cells to decitabine treatment. This study sheds light on the molecular mechanisms underlying the ASXL1-associated HMA resistance and proposes a promising therapeutic strategy for improving treatment outcomes in affected individuals.

Crocin enhances the sensitivity to paclitaxel in human breast cancer cells by reducing BIRC5 expression.

Paclitaxel (PTX) is one of the first-line chemotherapeutic agents for treating breast cancer. However, PTX resistance remains a major hurdle in breast cancer therapy. Crocin, the main chemical constituent of saffron, shows anti-cancer activity against various types of cancer. However, the effect of crocin on the resistance of PTX in breast cancer is still unknown. CCK-8 and TUNEL assays were employed to detect cell viability and apoptosis, respectively. The targets of crocin were predicted using HERB database and the targets associated with breast cancer were acquired using GEPIA database. The Venn diagram was utilized to identify the common targets between crocin and breast cancer. Baculoviral inhibitor of apoptosis repeat containing 5 (BIRC5) expression was detected by qRT-PCR and western blot analysis. The correlation between BIRC5 expression and survival was analyzed by Kaplan-Meier plotter and PrognoScan databases. Our data suggested that crocin aggravated PTX-induced decrease of viability and increase of apoptosis in MCF-7 and MCF-7/PTX cells. BIRC5 was identified as the target of crocin against breast cancer. Crocin inhibited BIRC5 expression in MCF-7 and MCF-7/PTX cells. BIRC5 is overexpressed in breast cancer tissues, as well as PTX-sensitive and PTX-resistant breast cancer cells. BIRC5 expression is related to the poor survival of patients with breast cancer. Depletion of BIRC5 strengthened PTX-induced viability reduction and promotion of apoptosis in MCF-7 and MCF-7/PTX cells. Moreover, BIRC5 overexpression reversed the inhibitory effect of crocin on PTX resistance in breast cancer cells. In conclusion, crocin enhanced the sensitivity of PTX in breast cancer cells partially through inhibiting BIRC5 expression.

Interaction of SMAC with a survivin-derived peptide alters essential cancer hallmarks: Tumor growth, inflammation, and immunosuppression.

Second mitochondrial-derived activator of caspase (SMAC), also known as direct inhibitor of apoptosis-binding proteins with low pI (Diablo), is known as a pro-apoptotic mitochondrial protein released into the cytosol in response to apoptotic signals. We recently reported SMAC overexpression in cancers as essential for cell proliferation and tumor growth due to non-apoptotic functions, including phospholipid synthesis regulation. These functions may be associated with its interactions with partner proteins. Using a peptide array with 768 peptides derived from 11 selected SMAC-interacting proteins, we identified SMAC-interacting sequences. These SMAC-binding sequences were produced as cell-penetrating peptides targeted to the cytosol, mitochondria, or nucleus, inhibiting cell proliferation and inducing apoptosis in several cell lines. For in vivo study, a survivin/baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5)-derived peptide was selected, due to its overexpression in many cancers and its involvement in mitosis, apoptosis, autophagy, cell proliferation, inflammation, and immune responses, as a target for cancer therapy. Specifically, a SMAC-targeting survivin/BIRC5-derived peptide, given intratumorally or intravenously, strongly inhibited lung tumor growth, cell proliferation, angiogenesis, and inflammation, induced apoptosis, and remodeled the tumor microenvironment. The peptide promoted tumor infiltration of CD-8+ cells and increased cell-intrinsic programmed cell death protein 1 (PD-1) and programmed cell death ligand 1 (PD-L1) expression, resulting in cancer cell self-destruction and increased tumor cell death, preserving immune cells. Thus, targeting the interaction between the multifunctional proteins SMAC and survivin represents an innovative therapeutic cancer paradigm.

Survivin inhibition ameliorates liver fibrosis by inducing hepatic stellate cell senescence and depleting hepatic macrophage population.

Persistent activation of hepatic stellate cells (HSCs) in the injured liver leads to the progression of liver injury from fibrosis to detrimental cirrhosis. In a previous study, we have shown that survivin protein is upregulated during the early activation of HSCs, which triggers the onset of liver fibrosis. However, the therapeutic potential of targeting survivin in a fully established fibrotic liver needs to be investigated. In this study, we chemically induced hepatic fibrosis in mice using carbon tetrachloride (CCl4) for 6 weeks, which was followed by treatment with a survivin suppressant (YM155). We also evaluated survivin expression in fibrotic human liver tissues, primary HSCs, and HSC cell line by histological analysis. αSMA+ HSCs in human and mice fibrotic liver tissues showed enhanced survivin expression, whereas the hepatocytes and quiescent (qHSCs) displayed minimal expression. Alternatively, activated M2 macrophage subtype induced survivin expression in HSCs through the TGF-ß-TGF-ß receptor-I/II signaling. Inhibition of survivin in HSCs promoted cell cycle arrest and senescence, which eventually suppressed their activation. In vivo, YM155 treatment increased the expression of cell senescence makers in HSCs around fibrotic septa such as p53, p21, and ß-galactosidase. YM155 treatment in vivo also reduced the hepatic macrophage population and inflammatory cytokine expression in the liver. In conclusion, downregulation of survivin in the fibrotic liver decreases HSC activation by inducing cellular senescence and modulating macrophage cytokine expression that collectively ameliorates liver fibrosis.

Downregulation of PTPRT elevates the expression of survivin and promotes the proliferation, migration, and invasion of lung adenocarcinoma.

BACKGROUND: Receptor-type tyrosine-protein phosphatase T (PTPRT) is a transmembrane protein that is involved in cell adhesion. We previously found that PTPRT was downregulated in multiple cancer types and the mutation of PTPRT was associated with cancer early metastasis. However, the impacts of PTPRT downregulation on tumour proliferation, invasion, and clinical interventions such as immune checkpoint inhibitor (ICI) therapies remained largely unknown. METHODS: Gene expression data of non-small cell lung cancer (NSCLC) samples from The Cancer Genome Atlas database were downloaded and used to detect the differential expressed genes between PTPRT-high and PTPRT-low subgroups. Knockdown and overexpress of PTPRT in lung cancer cell lines were performed to explore the function of PTPRT in vitro. Western blot and qRT-PCR were used to evaluate the expression of cell cycle-related genes. CCK-8 assays, wound-healing migration assay, transwell assay, and colony formation assay were performed to determine the functional impacts of PTPRT on cell proliferation, migration, and invasion. KM-plotter was used to explore the significance of selected genes on patient prognosis. RESULTS: PTPRT was found to be downregulated in tumours and lung cancer cell lines compared to normal samples. Cell cycle-related genes (BIRC5, OIP5, and CDCA3, etc.) were specifically upregulated in PTPRT-low lung adenocarcinoma (LUAD). Modulation of PTPRT expression in LUAD cell lines affected the expression of BIRC5 (survivin) significantly, as well as the proliferation, migration, and invasion of tumour cells. In addition, low PTPRT expression level was correlated with worse prognosis of lung cancer and several other cancer types. Furthermore, PTPRT downregulation was associated with elevated tumour mutation burden and tumour neoantigen burden in lung cancer, indicating the potential influence on tumour immunogenicity. CONCLUSION: Our findings uncovered the essential roles of PTPRT in the regulation of proliferation, migration, and invasion of LUAD, and highlighted the clinical significance of PTPRT downregulation in lung cancer.

17q Gain in Neuroblastoma: A Review of Clinical and Biological Implications.

Neuroblastoma (NB) is the most frequent extracranial solid childhood tumor. Despite advances in the understanding and treatment of this disease, the prognosis in cases of high-risk NB is still poor. 17q gain has been shown to be the most frequent genomic alteration in NB. However, the significance of this remains unclear because of its high frequency and association with other genetic modifications, particularly segmental chromosomal aberrations, 1p and 11q deletions, and MYCN amplification, all of which are also associated with a poor clinical prognosis. This work reviewed the evidence on the clinical and biological significance of 17q gain. It strongly supports the significance of 17q gain in the development of NB and its importance as a clinically relevant marker. However, it is crucial to distinguish between whole and partial chromosome 17q gains. The most important breakpoints appear to be at 17q12 and 17q21. The former distinguishes between whole and partial chromosome 17q gain; the latter is a site of IGF2BP1 and NME1 genes that appear to be the main oncogenes responsible for the functional effects of 17q gain.

Post-transcriptional regulation of BIRC5/survivin expression and induction of apoptosis in breast cancer cells by tristetraprolin.

Inhibition of apoptosis is one of the hallmarks of cancer and is a target of various therapeutic interventions. BIRC5 is an inhibitor of apoptosis that is aberrantly expressed in cancer leading to sustained growth of tumours. Post-transcriptional control mechanisms involving RNA-binding proteins and AU-rich elements (AREs) are fundamental to many cellular processes and changes in the expression or function of these proteins can promote an aberrant and pathological phenotype. BIRC5 mRNA has an ARE in its 3' UTR making it a candidate for regulation by the RNA binding proteins tristetraprolin (TTP) and HuR (ELAVL1). In this study, we investigated the binding of TTP and HuR by RNA-immunoprecipitation assays and found that these proteins were associated with BIRC5 mRNA to varying extents. Consequently, BIRC5 expression decreased when TTP was overexpressed and apoptosis was induced. In the absence of TTP, BIRC5 mRNA was stabilized, protein expression increased and the number of apoptotic cells declined. As an ARE-mRNA stabilizing protein, recombinant HuR led to upregulation of BIRC5 expression, whereas HuR silencing was concomitant with downregulation of BIRC5 mRNA and protein and increased cell death. Survival analyses demonstrated that increased TTP and low BIRC5 expression predicted an overall better prognosis compared to dysregulated TTP and high BIRC5. Thus, the results present a novel target of ARE-mediated post-transcriptional regulation.

BIRC5 facilitates cisplatin-chemoresistance in a m6A-dependent manner in ovarian cancer.

Cisplatin-based chemotherapy is the standard treatment for metastatic ovarian cancer (OC). However, chemoresistance continues to pose significant clinical challenges. Recent research has highlighted the baculoviral inhibitor of the apoptosis protein repeat-containing 5 (BIRC5) as a member of the inhibitor of the apoptosis protein (IAP) family. Notably, BIRC5, which has robust anti-apoptotic capabilities, is overexpressed in numerous cancers. Its dysfunction has been linked to challenges in cancer treatment. Yet, the role of BIRC5 in the chemoresistance of OC remains elusive. In our present study, we observed an upregulation of BIRC5 in cisplatin-resistant cell lines. This upregulation was associated with enhanced chemoresistance, which was diminished when the expression of BIRC5 was silenced. Intriguingly, BIRC5 exhibited a high number of N6-methyladenosine (m6A) binding sites. The modification of m6A was found to enhance the expression of BIRC5 by recognizing and binding to the 3'-UTR of mRNA. Additionally, the insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) was shown to stabilize BIRC5 mRNA, synergizing with METTL3 and intensifying chemoresistance. Supporting these in vitro findings, our in vivo experiments revealed that tumors were significantly smaller in size and volume when BIRC5 was silenced. This reduction was notably counteracted by co-silencing BIRC5 and overexpressing IGF2BP1. Our results underscored the pivotal role of BIRC5 in chemoresistance. The regulation of its expression and the stability of its mRNA were influenced by m6A modifications involving both METTL3 and IGF2BP1. These insights presented BIRC5 as a promising potential therapeutic target for addressing cisplatin resistance in OC.

Inhibition of Survivin Homodimerization Decreases Neuroblastoma Cell Growth.

Increased expression of BIRC5/survivin, a crucial regulator of the mitotic spindle checkpoint, is associated with poor prognosis in neuroblastoma (NB), the most common extracranial tumor of childhood. Transcriptional inhibitors of survivin have been tested in adult cancers and inhibitors of survivin homodimerization are emerging. We compared genetic inhibition of survivin transcription with the inhibition of survivin homodimerization by S12 and LQZ-7I, chosen from a larger panel of survivin dimerization inhibitors with activity against NB cells. Mice hemizygous for Birc5 were crossed with NB-prone TH-MYCN mice to generate Birc5+/-/MYCNtg/+ mice. The marked decrease of survivin transcription in these mice did not suffice to attenuate the aggressiveness of NB, even when tumors were transplanted into wild-type mice to assure that immune cell function was not compromised by the lack of survivin. In contrast, viability, clonogenicity and anchorage-independent growth of NB cells were markedly decreased by S12. S12 administered systemically to mice with subcutaneous NB xenotransplants decreased intratumoral hemorrhage, albeit not tumor growth. LQZ-7I, which directly targets the survivin dimerization interface, was efficacious in controlling NB cell growth in vitro at markedly lower concentrations compared to S12. LQZ-7I abrogated viability, clonogenicity and anchorage-independent growth, associated with massively distorted mitotic spindle formation. In vivo, LQZ-7I effectively reduced tumor size and cell proliferation of NB cells in CAM assays without apparent toxicity to the developing chick embryo. Collectively, these findings show that inhibiting survivin homodimerization with LQZ-7I holds promise for the treatment of NB and merits further investigation.

BIRC5 Gene Polymorphisms Are Associated with a Higher Stage of Local and Regional Disease in Oral and Oropharyngeal Squamous Cell Carcinomas.

Oral squamous cell carcinoma (OSCC) and oropharyngeal squamous cell carcinoma (OPSCC) are the most common types of cancers in the head and neck region (HNSCC). Despite very aggressive treatment modalities, the five-year survival rate has not changed for decades and is still around 60%. The search for potential specific biomarkers of aggressiveness or outcome indicators could be of great benefit in improving the treatment of these patients. One of the potential biomarkers is survivin, the protein product of the BIRC5 gene. In this study, we investigated the occurrence of BIRC5 gene polymorphisms in 48 patients with OSCC and OPSCC compared with healthy controls. A total of 18 polymorphisms were found, 11 of which occurred in HNSCC with a minor allele frequency (MAF) of more than 5%. Five polymorphisms (rs3764383, rs9904341, rs2071214, rs2239680, rs2661694) were significantly associated with tumor size, tumor stage, and advanced regional disease, but had no impact on survival.

Targeting survivin for cancer therapy: Strategies, small molecule inhibitors and vaccine based therapeutics in development.

Survivin is a member of the family of inhibitors of apoptosis proteins (IAPs). It is involved in the normal mitotic process and acts as an anti-apoptotic molecule. While terminally differentiated normal tissues lack survivin, several human malignancies have significant protein levels. Resistance to chemotherapy and radiation in tumor cells is associated with survivin expression. Decreased tumor development, apoptosis, and increased sensitivity to chemotherapy and radiation are all effects of downregulating survivin expression or activity. As a prospective cancer treatment, small molecules targeting the transcription and translation of survivin and molecules that can directly bind with the survivin are being explored both in pre-clinical and clinics. Pre-clinical investigations have found and demonstrated the effectiveness of several small-molecule survivin inhibitors. Unfortunately, these inhibitors have also been shown to have off-target effects, which could limit their clinical utility. In addition to small molecules, several survivin peptide vaccines are currently under development. These vaccines are designed to elicit a cytotoxic T-cell response against survivin, which could lead to the destruction of tumor cells expressing survivin. Some survivin-based vaccines are advancing through Phase II clinical studies. Overall, survivin is a promising cancer drug target. However, challenges still need to be addressed before the survivin targeted therapies can be widely used in the clinics.

Combined Inhibition of UBE2C and PLK1 Reduce Cell Proliferation and Arrest Cell Cycle by Affecting ACLY in Pan-Cancer.

Various studies have shown that the cell-cycle-related regulatory proteins UBE2C, PLK1, and BIRC5 promote cell proliferation and migration in different types of cancer. However, there is a lack of in-depth and systematic research on the mechanism of these three as therapeutic targets. In this study, we found a positive correlation between the expression of UBE2C and PLK1/BIRC5 in the Cancer Genome Atlas (TCGA) database, revealing a potential combination therapy candidate for pan-cancer. Quantitative real-time PCR (qRT-PCR), Western blotting (WB), cell phenotype detection, and RNA-seq techniques were used to evidence the effectiveness of the combination candidate. We found that combined interference of UBE2C with PLK1 and UBE2C with BIRC5 affected metabolic pathways by significantly downregulating the mRNA expression of IDH1 and ACLY, which was related to the synthesis of acetyl-CoA. By combining the PLK1 inhibitor volasertib and the ACLY inhibitor bempedoic acid, it showed a higher synergistic inhibition of cell viability and higher synergy scores in seven cell lines, compared with those of other combination treatments. Our study reveals the potential mechanisms through which cell-cycle-related genes regulate metabolism and proposes a potential combined targeted therapy for patients with higher PLK1 and ACLY expression in pan-cancer.

Designing Nanomedicines for Breast Cancer Therapy.

In 2020, breast cancer became the most diagnosed cancer worldwide. Conventional chemotherapies have major side effects due to their non-specific activities. Alternatively, short interfering RNA(siRNA)-carrying nanoparticles (NPs) have a high potential to overcome this non-specificity. Lipid-substituted polyethyleneimine (PEI) polymers (lipopolymers) have been reported as efficient non-viral carriers of siRNA. This study aims to engineer novel siRNA/lipopolymer nanocomplexes by incorporating anionic additives to obtain gene silencing through siRNA activity with minimal nonspecific toxicity. We first optimized our polyplexes in GFP+ MDA-MB-231 cells to effectively silence the GFP gene. Inclusion of phosphate buffer with pH 8.0 as complex preparation media and N-Lauroylsarcosine Sodium Salt as additive, achieved ~80% silencing with the least amount of undesired cytotoxicity, which was persistent for at least 6 days. The survivin gene was then selected as a target in MDA-MB-231 cells since there is no strong drug (i.e., small organic molecule) for inhibition of its oncogenic activity. The qRT-PCR, flow cytometry analysis and MTT assay revealed >80% silencing, ~95% cell uptake and >70% cell killing by the same formulation. We conclude that our lipopolymer can be further investigated as a lead non-viral carrier for breast cancer gene therapy.

BIRC5 Inhibition Is Associated with Pyroptotic Cell Death via Caspase3-GSDME Pathway in Lung Adenocarcinoma Cells.

Lung adenocarcinoma (LUAD) is a prevalent type of thoracic cancer with a poor prognosis and high mortality rate. However, the exact pathogenesis of this cancer is still not fully understood. One potential factor that can contribute to the development of lung adenocarcinoma is DNA methylation, which can cause changes in chromosome structure and potentially lead to the formation of tumors. The baculoviral IAP repeat containing the 5 (BIRC5) gene encodes the Survivin protein, which is a multifunctional gene involved in cell proliferation, migration, and invasion of tumor cells. This gene is elevated in various solid tumors, but its specific role and mechanism in lung adenocarcinoma are not well-known. To identify the potential biomarkers associated with lung adenocarcinoma, we screened the methylation-regulated differentially expressed genes (MeDEGs) of LUAD via bioinformatics analysis. Gene ontology (GO) process and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were applied to investigate the biological function and pathway of MeDEGs. A protein-protein interaction (PPI) network was employed to explore the key module and screen hub genes. We screened out eight hub genes whose products are aberrantly expressed, and whose DNA methylation modification level is significantly changed in lung adenocarcinoma. BIRC5 is a bona fide marker which was remarkably up-regulated in tumor tissues. Flow cytometry analysis, lactate dehydrogenase release (LDH) assay and Micro-PET imaging were performed in A549 cells and a mouse xenograft tumor to explore the function of BIRC5 in cell death of lung adenocarcinoma. We found that BIRC5 was up-regulated and related to a high mortality rate in lung adenocarcinoma patients. Mechanically, the knockdown of BIRC5 inhibited the proliferation of A549 cells and induced pyroptosis via caspase3/GSDME signaling. Our findings have unraveled that BIRC5 holds promise as a novel biomarker and therapeutic target for lung adenocarcinoma. Additionally, we have discovered a novel pathway in which BIRC5 inhibition can induce pyroptosis through the caspase3-GSDME pathway in lung adenocarcinoma cells.

Downregulated BIRC5 inhibits proliferation and metastasis of melanoma through the ß-catenin/HIF-1α/VEGF/MMPs pathway.

PURPOSE: Melanoma is a malignant skin tumor caused by melanocytes and associated with high mortality rates. This study aims to investigate the specific mechanism of ZWZ-3 in melanoma proliferation and metastasis. METHODS: RNA sequencing was performed to identify the effect of ZWZ-3 on gene expression. siRNA was used to inhibit BIRC5 gene expression in the B16F10 cell line. A zebrafish tumor model was used to assess the therapeutic effect of ZWZ-3 in vivo. Mechanistic insights into the inhibition of tumor metastasis by ZWZ-3 were obtained through analysis of tumor tissue sections in mice. RESULTS: Our findings demonstrated that ZWZ-3 suppressed melanoma cell proliferation and migration. We performed RNA sequencing in melanoma cells after the treatment with ZWZ-3 and found that Birc5, which is closely associated with tumor metastasis, was significantly down-regulated. Bioinformatics analysis and the immuno-histochemical results of tissue chips for melanoma further confirmed the high expression of BIRC5 in melanoma and its effect on disease progression. Moreover, Birc5 knock-down significantly inhibited melanoma cell proliferation and metastasis, which was correlated with the ß-catenin/HIF-1α/VEGF/MMPs pathway. Additionally, ZWZ-3 significantly inhibited tumor growth in the zebrafish tumor model without any evident side effects. Histological and immuno-histochemical analyses revealed that ZWZ-3 inhibited tumor cell metastasis by down-regulating HIF-1α, VEGF, and MMP9. CONCLUSION: Our findings revealed that ZWZ-3 could downregulate BIRC5 and inhibit melanoma proliferation and metastasis through the ß-catenin/HIF-1α/VEGF/MMPs pathway. Therefore, BIRC5 represents a promising therapeutic target for the treatment of melanoma.

A co-formulation of interferons alpha2b and gamma distinctively targets cell cycle in the glioblastoma-derived cell line U-87MG.

BACKGROUND: HeberFERON is a co-formulation of α2b and γ interferons, based on their synergism, which has shown its clinical superiority over individual interferons in basal cell carcinomas. In glioblastoma (GBM), HeberFERON has displayed promising preclinical and clinical results. This led us to design a microarray experiment aimed at identifying the molecular mechanisms involved in the distinctive effect of HeberFERON compared to the individual interferons in U-87MG model. METHODS: Transcriptional expression profiling including a control (untreated) and three groups receiving α2b-interferon, γ-interferon and HeberFERON was performed using an Illumina HT-12 microarray platform. Unsupervised methods for gene and sample grouping, identification of differentially expressed genes, functional enrichment and network analysis computational biology methods were applied to identify distinctive transcription patterns of HeberFERON. Validation of most representative genes was performed by qPCR. For the cell cycle analysis of cells treated with HeberFERON for 24 h, 48 and 72 h we used flow cytometry. RESULTS: The three treatments show different behavior based on the gene expression profiles. The enrichment analysis identified several mitotic cell cycle related events, in particular from prometaphase to anaphase, which are exclusively targeted by HeberFERON. The FOXM1 transcription factor network that is involved in several cell cycle phases and is highly expressed in GBMs, is significantly down regulated. Flow cytometry experiments corroborated the action of HeberFERON on the cell cycle in a dose and time dependent manner with a clear cellular arrest as of 24 h post-treatment. Despite the fact that p53 was not down-regulated, several genes involved in its regulatory activity were functionally enriched. Network analysis also revealed a strong relationship of p53 with genes targeted by HeberFERON. We propose a mechanistic model to explain this distinctive action, based on the simultaneous activation of PKR and ATF3, p53 phosphorylation changes, as well as its reduced MDM2 mediated ubiquitination and export from the nucleus to the cytoplasm. PLK1, AURKB, BIRC5 and CCNB1 genes, all regulated by FOXM1, also play central roles in this model. These and other interactions could explain a G2/M arrest and the effect of HeberFERON on the proliferation of U-87MG. CONCLUSIONS: We proposed molecular mechanisms underlying the distinctive behavior of HeberFERON compared to the treatments with the individual interferons in U-87MG model, where cell cycle related events were highly relevant.