GINS2 regulates temozolomide chemosensitivity via the EGR1/ECT2 axis in gliomas.

Temozolomide (TMZ), a DNA alkylating agent, has become the primary treatment for glioma, the most common malignancy of the central nervous system. Although TMZ-containing regimens produce significant clinical response rates, some patients inevitably suffer from inferior treatment outcomes or disease relapse, likely because of poor chemosensitivity of glioma cells due to a robust DNA damage response (DDR). GINS2, a subunit of DNA helicase, contributes to maintaining genomic stability and is highly expressed in various cancers, promoting their development. Here, we report that GINS2 was upregulated in TMZ-treated glioma cells and co-localized with γH2AX, indicating its participation in TMZ-induced DDR. Furthermore, GINS2 regulated the malignant phenotype and TMZ sensitivity of glioma cells, mostly by promoting DNA damage repair by affecting the mRNA stability of early growth response factor 1 (EGR1), which in turn regulates the transcription of epithelial cell-transforming sequence 2 (ECT2). We constructed a GINS2-EGR1-ECT2 prognostic model, which accurately predicted patient survival. Further, we screened Palbociclib/BIX-02189 which dampens GINS2 expression and synergistically inhibits glioma cell proliferation with TMZ. These findings delineate a novel mechanism by which GINS2 regulates the TMZ sensitivity of glioma cells and propose a promising combination therapy to treat glioma.

Mechanisms of Resistance to Tyrosine Kinase Inhibitors in ROS1 Fusion-Positive Nonsmall Cell Lung Cancer.

BACKGROUND: ROS1 fusion-positive (ROS1+) nonsmall cell lung cancer (NSCLC) patients are highly sensitive to tyrosine kinase inhibitor (TKI) treatments. However, acquired TKI resistance remains the major hurdle preventing patients from experiencing prolonged benefits. METHODS: 107 advanced or metastatic ROS1+ NSCLC patients who progressed on crizotinib and lorlatinib were recruited. Tissue and plasma samples were collected at baseline (N = 50), postcrizotinib (N = 91), and postlorlatinib (N = 21), which were all subject to the 139-gene targeted next-generation DNA sequencing. Molecular dynamics modeling was performed to investigate the effects of ROS1 mutations on binding to different TKIs. RESULTS: In patients with postcrizotinib and postlorlatinib samples, an accumulation of on- and off-target resistance alterations after multiple TKI treatments was observed. ROS1 G2032R and MET amplification were the most common on-target and off-target alterations, respectively. Patients with CD74-ROS1 and SLC34A2-ROS1 had longer progression-free survival (PFS) (P < 0.001) and higher rates of resistance mutations (on-target, P = 0.001; off-target, P = 0.077) than other ROS1 fusion variants following crizotinib treatment. Ten distinct on-target resistance mutations were detected after TKI therapies, of which 4 were previously unreported (ROS1 L2010M, G1957A, D1988N, L1982V). Molecular dynamics simulations showed that all 4 mutations were refractory to crizotinib, while G1957A, D1988N, and L1982V were potentially sensitive to lorlatinib and entrectinib. CONCLUSIONS: This study provided a comprehensive portrait of TKI-resistance mechanisms in ROS1+ NSCLC patients. Using in silico simulations of TKI activity, novel secondary mutations that may confer TKI resistance were identified and may support clinical therapeutic decision-making.

B-cell lymphoma-2 family proteins in the crosshairs: Small molecule inhibitors and activators for cancer therapy.

The B-cell lymphoma-2 (BCL-2) family of proteins plays a crucial role in the regulation of apoptosis, offering a dual mechanism for its control. Numerous studies have established a strong association between gene disorders of these proteins and the proliferation of diverse cancer cell types. Consequently, the identification and development of drugs targeting BCL-2 family proteins have emerged as a prominent area in antitumor therapy. Over the last two decades, several small-molecules have been designed to modulate the protein-protein interactions between anti- and proapoptotic BCL-2 proteins, effectively suppressing tumor growth and metastasis in vivo. The primary focus of research has been on developing BCL-2 homology 3 (BH3) mimetics to target antiapoptotic BCL-2 proteins, thereby competitively releasing proapoptotic BCL-2 proteins and restoring the blocked intrinsic apoptotic program. Additionally, for proapoptotic BCL-2 proteins, exogenous small molecules have been explored to activate cell apoptosis by directly interacting with executioner proteins such as BCL-2-associated X protein (BAX) or BCL-2 homologous antagonist/killer protein (BAK). In this comprehensive review, we summarize the inhibitors and activators (sensitizers) of BCL-2 family proteins developed over the past decades, highlighting their discovery, optimization, preclinical and clinical status, and providing an overall landscape of drug development targeting these proteins for therapeutic purposes.

MIG6 Mediates Adaptive and Acquired Resistance to ALK/ROS1 Fusion Kinase Inhibition through EGFR Bypass Signaling.

Despite the initial benefit from tyrosine kinase inhibitors (TKI) targeting oncogenic ALK and ROS1 gene fusions in non-small cell lung cancer, complete responses are rare and resistance ultimately emerges from residual tumor cells. Although several acquired resistance mechanisms have been reported at the time of disease progression, adaptative resistance mechanisms that contribute to residual diseases before the outgrowth of tumor cells with acquired resistance are less clear. For the patients who have progressed after TKI treatments, but do not demonstrate ALK/ROS1 kinase mutations, there is a lack of biomarkers to guide effective treatments. Herein, we found that phosphorylation of MIG6, encoded by the ERRFI1 gene, was downregulated by ALK/ROS1 inhibitors as were mRNA levels, thus potentiating EGFR activity to support cell survival as an adaptive resistance mechanism. MIG6 downregulation was sustained following chronic exposure to ALK/ROS1 inhibitors to support the establishment of acquired resistance. A higher ratio of EGFR to MIG6 expression was found in ALK TKI-treated and ALK TKI-resistant tumors and correlated with the poor responsiveness to ALK/ROS1 inhibition in patient-derived cell lines. Furthermore, we identified and validated a MIG6 EGFR-binding domain truncation mutation in mediating resistance to ROS1 inhibitors but sensitivity to EGFR inhibitors. A MIG6 deletion was also found in a patient after progressing to ROS1 inhibition. Collectively, this study identifies MIG6 as a novel regulator for EGFR-mediated adaptive and acquired resistance to ALK/ROS1 inhibitors and suggests EGFR to MIG6 ratios and MIG6-damaging alterations as biomarkers to predict responsiveness to ALK/ROS1 and EGFR inhibitors.

Parkin enhances sensitivity of paclitaxel to nasopharyngeal carcinoma by activating BNIP3/NIX-mediated mitochondrial autophagy.

As a malignant head and neck cancer, nasopharyngeal carcinoma (NPC) has high morbidity. Parkin expression has been reported to be reduced in NPC tissues and its upregulation could enhance paclitaxel-resistant cell cycle arrest. This study was performed to explore the possible mechanism of Parkin related to B-cell lymphoma-2 (Bcl-2)/adenovirus E1B 19 kDa interacting protein 3 (BNIP3)/BNIP3-like (NIX)-mediated mitochondrial autophagy in NPC cells. Initially, after Parkin overexpression or silencing, cell viability and proliferation were evaluated by lactate dehydrogenase and colony formation assays. JC-1 staining was used to assess the mitochondrial membrane potential. In addition, the levels of cellular reactive oxygen species (ROS) and mitochondrial ROS were detected using DCFH-DA staining and mitochondrial ROS (MitoSOX) red staining. The expression of proteins was measured using Western blot. Results showed that Parkin overexpression inhibited, whereas Parkin knockdown promoted the proliferation of paclitaxel-treated NPC cells. Besides, Parkin overexpression induced, whereas Parkin knockdown inhibited mitochondrial apoptosis in paclitaxel-treated NPC cells, as evidenced by the changes of Cytochrome C (mitochondria), Cytochrome C (cytoplasm), BAK, and Bcl-2 expression. Moreover, the levels of ROS, mitochondrial membrane potential, and LC3II/LC3I in paclitaxel-treated C666-1 cells were hugely elevated by Parkin overexpression and were all declined by Parkin knockdown in CNE-3 cells. Furthermore, Parkin upregulation activated, whereas Parkin downregulation inactivated BNIP3/NIX signaling. Further, BNIP3 silencing or overexpression reversed the impacts of Parkin upregulation or downregulation on the proliferation and mitochondrial apoptosis of paclitaxel-treated NPC cells. Particularly, Mdivi-1 (mitophagy inhibitor) or rapamycin (an activator of autophagy) exerted the same effects on NPC cells as BNIP3 silencing or overexpression, respectively. Collectively, Parkin overexpression activated BNIP3/NIX-mediated mitochondrial autophagy to enhance sensitivity to paclitaxel in NPC.

PARP inhibitor exerts an anti-tumor effect via LMO2 and synergizes with cisplatin in natural killer/T cell lymphoma.

BACKGROUND: PARP inhibitor (PARPi), as a kind of DNA damage repair inhibitor, has been shown to be effective in various solid tumors and hematologic malignancies. Natural killer/T cell lymphoma (NKTCL) is a highly aggressive malignancy, the treatment of which has long been a major challenge in the clinic. Here, we investigated the efficacy and mechanism of PARPi, and the therapeutic value of PARPi combined with cisplatin in NKTCL. METHODS: The cell proliferation, cell apoptosis, and cell cycle of NKTCL cells were detected respectively by CCK-8 and flow cytometry. The changes of mRNA expression and protein level were measured respectively by mRNA-sequencing, quantitative real-time PCR, western blotting, and immunofluorescence. LMO2 expression was detected by immunohistochemistry and western blotting. Targeted knockdown of LMO2 was conducted by short hairpin RNA. The tumor xenograft models were established to evaluate the efficacy of drugs in vivo. RESULTS: PARPi inhibited cell proliferation, promoted cell apoptosis, and induced S-phase cell cycle arrest in NKTCL cells. PARPi led to the accumulation of DNA damage by blocking DNA repair and DNA replication. Additionally, LMO2 deficiency reduced the sensitivity of NKTCL cells to PARPi. Finally, the combination of PARPi and cisplatin exhibited significant synergistic effects both in vitro and in vivo. CONCLUSIONS: In summary, we found that PARPi exerted an anti-tumor effect via LMO2 and synergized with cisplatin in NKTCL, which provides the theoretical basis for the clinical application of PARPi.

Protective effects of melatonin on myocardial microvascular endothelial cell injury under hypertensive state by regulating Mst1.

BACKGROUND: This study explored the protective effects of melatonin on the hypertensive model in myocardial microvascular endothelial cells. METHODS: Mouse myocardial microvascular endothelial cells were intervened with angiotensin II to establish hypertensive cell model and divided into control, hypertension (HP), hypertension + adenovirus negative control (HP + Ad-NC), hypertension + adenovirus carrying Mst1 (HP + Ad-Mst1), hypertension + melatonin (HP + MT), hypertension + adenovirus negative control + melatonin (HP + Ad-NC + MT), and hypertension + adenovirus carrying Mst1 + melatonin (HP + Ad-Mst1 + MT) groups. Autophagosomes were observed by transmission electron microscope. Mitochondrial membrane potential was detected by JC-1 staining. Apoptosis was detected by flow cytometry. Oxidative stress markers of MDA, SOD and GSH-PX were measured. The expression of LC3 and p62 was detected by immunofluorescence. Expression levels of Mst1, p-Mst1, Beclin1, LC3, and P62 were detected with Western blot. RESULTS: Compared with the control group, the autophagosomes in HP, HP + Ad-Mst1, and HP + Ad-NC groups were significantly reduced. Compared with HP group, the autophagosomes in HP + Ad-Mst1 group were significantly reduced. The apoptosis of HP + MT group was significantly lower than HP group. Compared with HP + Ad-Mst1 group, the apoptosis of HP + Ad-Mst1 + MT group was significantly reduced. The ratio of JC-1 monomer in HP + MT group was significantly lower than HP group. Compared with HP + Ad-Mst1 group, the mitochondrial membrane potential of HP + Ad-Mst1 + MT group was also significantly reduced. MDA content in HP + MT group was significantly reduced, but SOD and GSH-PX activities were significantly increased. Compared with HP + Ad-Mst1 group, MDA content in HP + Ad-Mst1 + MT group was significantly reduced, whereas SOD and GSH-PX activities were increased significantly. Mst1 and p-Mst1 proteins in HP + MT group were significantly reduced. Compared with HP + Ad-Mst1 group, Mst1 and p-Mst1 in HP + Ad-Mst1 + MT group were reduced. P62 level was significantly decreased, while Beclin1 and LC3II levels were significantly increased. P62 in HP + MT group was significantly reduced, while Beclin1 and LC3II were significantly increased. Compared with HP + Ad-Mst1 group, P62 in HP + Ad-Mst1 + MT group was significantly reduced, but Beclin1 and LC3II were significantly increased. CONCLUSION: Melatonin may inhibit apoptosis, increase mitochondrial membrane potential, and increase autophagy of myocardial microvascular endothelial cells under hypertensive state via inhibiting Mst1 expression, thereby exerting myocardial protective effect.

PBX1-promoted SFRP4 transcription inhibits cell proliferation and epithelial-mesenchymal transition in endometrial carcinoma.

OBJECTIVE: To explore the effects and mechanisms of action of the PBX1/secreted frizzled-related protein 4 (SFRP4) axis in endometrial carcinoma (EC). METHODS: The expression of PBX1 and SFRP4 was analyzed using bioinformatics prediction, followed by validation in EC cells using quantitative reverse transcription-polymerase chain reaction and western blotting. After transduction with overexpression vectors for PBX1 and SFRP4, migration, proliferation, and invasion of EC cells were measured, accompanied by the detection of E-cadherin, Snail, N-cadherin, Vimentin, ß-catenin, GSK-3ß, and C-myc expression. The association between PBX1 and SFRP4 was validated using dual luciferase reporter gene and chromatin immunoprecipitation assays. RESULTS: PBX1 and SFRP4 were downregulated in EC cells. Overexpression of PBX1 or SFRP4 resulted in weakened cell proliferation, migration, and invasion, as well as decreased expression of Snail, N-cadherin, Vimentin, ß-catenin, GSK-3ß, and C-myc and increased expression of E-cadherin. PBX1 bound to the SFRP4 promoter and promoted its transcription. Knockdown of SFRP4 reversed the repression of overexpressed PBX1 in the malignant phenotypes and EMT of EC cells, and PBX1 repressed Wnt/ß-catenin pathway activation by upregulating SFRP4 transcription. CONCLUSION: PBX1 inhibited activation of the Wnt/ß-catenin pathway by promoting SFRP4 transcription, thereby suppressing malignant phenotypes in EC cells and the EMT process.

Long-lasting memory of jasmonic acid-dependent immunity requires DNA demethylation and ARGONAUTE1.

Stress can have long-lasting impacts on plants. Here we report the long-term effects of the stress hormone jasmonic acid (JA) on the defence phenotype, transcriptome and DNA methylome of Arabidopsis. Three weeks after transient JA signalling, 5-week-old plants retained induced resistance (IR) against herbivory but showed increased susceptibility to pathogens. Transcriptome analysis revealed long-term priming and/or upregulation of JA-dependent defence genes but repression of ethylene- and salicylic acid-dependent genes. Long-term JA-IR was associated with shifts in glucosinolate composition and required MYC2/3/4 transcription factors, RNA-directed DNA methylation, the DNA demethylase ROS1 and the small RNA (sRNA)-binding protein AGO1. Although methylome analysis did not reveal consistent changes in DNA methylation near MYC2/3/4-controlled genes, JA-treated plants were specifically enriched with hypomethylated ATREP2 transposable elements (TEs). Epigenomic characterization of mutants and transgenic lines revealed that ATREP2 TEs are regulated by RdDM and ROS1 and produce 21 nt sRNAs that bind to nuclear AGO1. Since ATREP2 TEs are enriched with sequences from IR-related defence genes, our results suggest that AGO1-associated sRNAs from hypomethylated ATREP2 TEs trans-regulate long-lasting memory of JA-dependent immunity.

MicroRNA miR-34a-5p inhibition restrains oxidative stress injury of macrophages by targeting MDM4.

OBJECTIVE: Atherosclerosis is a chronic cardiovascular disease associated with oxidative stress damage, which is caused by excessive oxidation of low-density lipoprotein (ox-LDL). The role of microRNA miR-34a-5p on oxidative stress in ox-LDL-treated macrophages was investigated in this study. METHODS: Flow cytometry was prepared for assessing THP1-derived macrophage apoptosis. The protein and expression levels of miR-34a-5p and MDM4 were examined by Western blot and RT-qPCR, respectively. We also measured the levels of total cholesterol (TC) and triglyceride to determine the lipid accumulation. Subsequently, the activities of superoxide dismutase, malondialdehyde, and reactive oxygen species revealed the level of oxidative stress injury after miR-34a-5p and MDM4 knockdown. RESULTS: After ox-LDL treatment, cell apoptosis of macrophages increased in a dose-dependent and time-dependent manner. With the increase of ox-LDL treatment and the prolongation of treatment time, the expression level of miR-34a-5p was upregulated. Next, interfering with miR-34a-5p inhibited lipid accumulation and oxidative stress injury in ox-LDL-stimulated macrophages. MDM4 was a target gene of miR-34a-5p and was upregulated in ox-LDL-stimulated macrophages. With the increase of ox-LDL treatment and the prolongation of treatment time, the expression level of MDM4 was downregulated. Importantly, MDM4 knockdown partially counteracted the inhibitory effect of miR-34a-5p on oxidative stress injury. CONCLUSION: MicroRNA miR-34a-5p knockdown suppressed oxidative stress injury via MDM4 in ox-LDL-treated macrophages.

A postpartum enriched environment rescues impaired cognition and oxidative markers in aged mice with gestational inflammation.

INTRODUCTION: Previous studies have shown that gestational inflammation can accelerate age-associated cognitive decline (AACD) in maternal mice; enriched environments (EEs) have been reported to protect normally aging mice from AACD and improve mitochondrial function. However, it is unclear whether the nitrosative stress-related proteins tet methylcytosine dioxygenase 1 (TET1) and S-nitrosoglutathione reductase (GSNOR) are involved in the accelerated aging process of gestational inflammation and whether EEs can slow this process. METHODS: In this study, CD-1 female mice on the 15th day of pregnancy were injected with bacterial lipopolysaccharide (50 µg/kg; LPS group) or an equivalent amount of normal saline (CON group) from the abdominal cavity for 4 consecutive days. Twenty-one days after delivery, half of the LPS-treated mice were randomly selected for EE until the end of the behavioral experiment (LPS-E group). When the female rats were raised to 6 months and 18 months of age, the Morris water maze (MWM) was used to detect spatial learning and memory ability; RT-PCR and Western blots were used to measure the mRNA and protein levels of hippocampal TET1 and GSNOR. RESULTS: As for the control group, compared with 6-month-old mice, the spatial learning and memory ability of 18-month-old mice decreased, and the hippocampal TET1 and GSNOR mRNA and protein levels were decreased. Gestational inflammation exacerbated these age-related changes, but an EE alleviated the effects. Pearson's correlation analysis indicated that performance during the learning and memory periods in the MWM correlated with the levels of hippocampal TET1 and GSNOR. CONCLUSIONS: Our findings suggest that gestational inflammation accelerates age-related learning and memory impairments and that postpartum EE exposure could alleviate these changes. These effects may be related to hippocampal TET1 and GSNOR expression.

Inhibition of MCL1 induces apoptosis in anaplastic large cell lymphoma and in primary effusion lymphoma.

Overexpression of antiapoptotic BCL2 family proteins occurs in various hematologic malignancies and contributes to tumorigenesis by inhibiting the apoptotic machinery of the cells. Antagonizing BH3 mimetics provide an option for medication, with venetoclax as the first drug applied for chronic lymphocytic leukemia and for acute myeloid leukemia. To find additional hematologic entities with ectopic expression of BCL2 family members, we performed expression screening of cell lines applying the LL-100 panel. Anaplastic large cell lymphoma (ALCL) and primary effusion lymphoma (PEL), 2/22 entities covered by this panel, stood out by high expression of MCL1 and low expression of BCL2. The MCL1 inhibitor AZD-5991 induced apoptosis in cell lines from both malignancies, suggesting that this BH3 mimetic might be efficient as drug for these diseases. The ALCL cell lines also expressed BCLXL and BCL2A1, both contributing to survival of the cells. The combination of specific BH3 mimetics yielded synergistic effects, pointing to a novel strategy for the treatment of ALCL. The PI3K/mTOR inhibitor BEZ-235 could also efficiently be applied in combination with AZD-5991, offering an alternative to avoid thrombocytopenia which is associated with the use of BCLXL inhibitors.

Selective BH3 mimetics synergize with BET inhibition to induce mitochondrial apoptosis in rhabdomyosarcoma cells.

BH3 mimetics are promising novel anticancer therapeutics. By selectively inhibiting BCL-2, BCL-xL, or MCL-1 (i.e. ABT-199, A-1331852, S63845) they shift the balance of pro- and anti-apoptotic proteins in favor of apoptosis. As Bromodomain and Extra Terminal (BET) protein inhibitors promote pro-apoptotic rebalancing, we evaluated the potential of the BET inhibitor JQ1 in combination with ABT-199, A-1331852 or S63845 in rhabdomyosarcoma (RMS) cells. The strongest synergistic interaction was identified for JQ1/A-1331852 and JQ1/S63845 co-treatment, which reduced cell viability and long-term clonogenic survival. Mechanistic studies revealed that JQ1 upregulated BIM and NOXA accompanied by downregulation of BCL-xL, promoting pro-apoptotic rebalancing of BCL-2 proteins. JQ1/A-1331852 and JQ1/S63845 co-treatment enhanced this pro-apoptotic rebalancing and triggered BAK- and BAX-dependent apoptosis since a) genetic silencing of BIM, BAK or BAX, b) inhibition of caspase activity with zVAD.fmk and c) overexpression of BCL-2 all rescued JQ1/A-1331852- and JQ1/S63845-induced cell death. Interestingly, NOXA played a different role in both treatments, as genetic silencing of NOXA significantly rescued from JQ1/A-1331852-mediated apoptosis but not from JQ1/S63845-mediated apoptosis. In summary, JQ1/A-1331852 and JQ1/S63845 co-treatment represent new promising therapeutic strategies to synergistically trigger mitochondrial apoptosis in RMS.

AXL Inhibitor TP-0903 Reduces Metastasis and Therapy Resistance in Pancreatic Cancer.

Pancreatic cancer is the third leading cause of cancer-related deaths in the United States with a 5-year survival less than 5%. Resistance to standard therapy and limited response to immune checkpoint blockade due to the immunosuppressive and stroma-rich microenvironment remain major challenges in the treatment of pancreatic cancer. A key cellular program involved in therapy resistance is epithelial plasticity, which is also associated with invasion, metastasis, and evasion of immune surveillance. The receptor tyrosine kinase AXL is a key driver of tumor cell epithelial plasticity. High expression and activity of AXL is associated with poor prognosis, metastasis, and therapy resistance in multiple types of cancer including pancreatic. Here, we show that an AXL inhibitor (TP-0903), has antitumor and therapy sensitizing effects in preclinical models of pancreatic ductal adenocarcinoma (PDA). We demonstrate that TP-0903 as a single agent or in combination with gemcitabine and/or anti-programmed cell death protein 1 (PD1) antibody has anti-metastatic and anti-tumor effects in PDA tumor bearing mice, leading to increased survival. In addition, gene expression analysis of tumors demonstrated upregulation of pro-inflammatory and immune activation genes in tumors from TP-0903-treated animals compared with the vehicle, indicating pharmacologic inhibition of AXL activation leads to an immunostimulatory microenvironment. This effect was augmented when TP-0903 was combined with gemcitabine and anti-PD1 antibody. These results provide clear rationale for evaluating TP-0903 in the treatment of pancreatic cancer.

Netrin-like domain of sFRP4, a Wnt antagonist inhibits stemness, metastatic and invasive properties by specifically blocking MMP-2 in cancer stem cells from human glioma cell line U87MG.

Rapid proliferation, high stemness potential, high invasiveness and apoptotic evasion are the distinctive hallmarks of glioma malignancy. The dysregulation of the Wnt/ß-catenin pathway is the key factor regulating glioma malignancy. Wnt antagonist, secreted frizzled-related protein 4 (sFRP4), which has a prominent pro-apoptotic role in glioma stem cells, has two functional domains, the netrin-like domain (NLD), and cysteine-rich domain (CRD) both of which contribute to apoptotic properties of the whole protein. However, there are no reports elucidating the specific effects of individual domains of sFRP4 in inhibiting the invasive properties of glioma. This study explores the efficacy of the domains of sFRP4 in inhibiting the key hallmarks of glioblastoma such as invasion, metastasis, and stemness. We overexpressed sFRP4 and its domains in the glioblastoma cell line, U87MG cells and observed that both CRD and NLD domains played prominent roles in attenuating cancer stem cell properties. Significantly, we could demonstrate for the first time that both NLD and CRD domains negatively impacted the key driver of metastasis and migration, the matrix metalloproteinase-2 (MMP-2). Mechanistically, compared to CRD, NLD domain suppressed MMP-2 mediated invasion more effectively in glioma cells as observed in matrigel invasion assay and a function-blocking antibody assay. Fluorescent matrix degradation assay further revealed that NLD reduces matrix degradation. NLD also significantly disrupted fibronectin assembly and decreased cell adhesion in another glioma cell line LN229. In conclusion, the NLD peptide of sFRP4 could be a potent short peptide therapeutic candidate for targeting MMP-2-mediated invasion in the highly malignant glioblastoma multiforme.

Therapeutic targeting of SPIB/SPI1-facilitated interplay of cancer cells and neutrophils inhibits aerobic glycolysis and cancer progression.

BACKGROUND: As a metabolic reprogramming feature, cancer cells derive most of their energy from aerobic glycolysis, while its regulatory mechanisms and therapeutic strategies continue to be illusive. METHODS: Integrative analysis of publically available expression profile datasets was used to identify critical transcriptional regulators and their target glycolytic enzymes. The functions and acting mechanisms of transcriptional regulators in cancer cells were investigated by using in vitro and in vivo assays. The Kaplan-Meier curve and log-rank assay were used to conduct the survival study. RESULTS: Salmonella pathogenicity island 1 (SPI1/PU.1), a haematopoietic transcription factor, was identified to facilitate glycolytic process, tumourigenesis, invasiveness, as well as metastasis of colon cancer cells, which was interplayed by tumour-associated neutrophils. Mechanistically, neutrophils delivered SPI1 mRNA via extracellular vesicles, resulting in enhanced SPI1 expression of cancer cells. Through physical interaction with SPI1-related protein (SPIB), SPI1 drove expression of glycolytic genes within cancer cells, which in turn induced polarization of neutrophils via glycolytic metabolite lactate. Depletion of neutrophils or SPIB-SPI1 interaction in cancer cells significantly inhibited glycolytic process, tumourigenesis and aggressiveness. Upregulation of SPI1 or SPIB was found to be associated with poor prognosis in patients suffering from colon cancer. CONCLUSIONS: Therapeutic targeting of SPIB/SPI1-facilitated interplay of cancerous cells and neutrophils suppresses aerobic glycolysis and progression of cancer.

Assembling BH3-mimic peptide into a nanocluster to target intracellular Bcl2 towards the apoptosis induction of cancer cell.

Bcl-2, an anti-apoptotic protein, is always overexpressed in tumor cells to suppress the pro-apoptotic function of Bax, thereby prolonging the life of the tumor. However, BH3 proteins could directly activate Bax via antagonizing Bcl-2 to induce apoptosis in response to the stimulation. Thus, mimicking BH3 proteins with a peptide is a potential strategy for anti-cancer therapy. Unfortunately, clinical translation of BH3-mimic peptide is hindered by its inefficacious cellular internalization and proteolysis resistance. Herein, we translated a BH3-mimic peptide into a peptide-auric spheroidal nanocluster (BH3-AuNp), in which polymeric BH3-Auric precursors [Au1+-S-BH3]narein situself-assembled on the surface of gold nanoparticles by a one-pot synthesis. Expectedly, this strategy could improve the anti-proteolytic ability and cytomembrane penetrability of the BH3 peptide. As a result, BH3-AuNp successfully induced the apoptosis of two cancer cell lines by an order of magnitude compared to BH3. This therapeutic and feasible peptide nano-engineering strategy will help peptides overcome the pharmaceutical obstacles, awaken its biological functions, and possibly revive the research about peptide-derived nanomedicine.

Dynamic BH3 profiling method for rapid identification of active therapy in BH3 mimetics resistant xenograft mouse models.

The clinical effectiveness of BH3 mimetics therapy is limited by the inevitable emergence of acquired resistance. We present a protocol to model in vivo acquired resistance to BH3 mimetics in patient-derived xenograft (PDX) mouse models of acute myeloid leukemia. Using resistant PDXs as a valuable model, we next introduce a protocol for dynamic BH3 profiling (DBP) method. DBP allows functional identification of effective drug therapies based on measurements of drug-induced apoptosis signaling to overcome in vivo BH3 mimetics resistance. For complete details on the use and execution of this protocol, please refer to Bhatt et al. (2020).

Design, Synthesis, and Structural Characterization of Lysine Covalent BH3 Peptides Targeting Mcl-1.

Modulating disease-relevant protein-protein interactions (PPIs) using pharmacological tools is a critical step toward the design of novel therapeutic strategies. Over the years, however, targeting PPIs has proven a very challenging task owing to the large interfacial areas. Our recent efforts identified possible novel routes for the design of potent and selective inhibitors of PPIs using a structure-based design of covalent inhibitors targeting Lys residues. In this present study, we report on the design, synthesis, and characterizations of the first Lys-covalent BH3 peptide that has a remarkable affinity and selectivity for hMcl-1 over the closely related hBfl-1 protein. Our structural studies, aided by X-ray crystallography, provide atomic-level details of the inhibitor interactions that can be used to further translate these discoveries into novel generation, Lys-covalent pro-apoptotic agents.

CircTET1 Inhibits Retinoblastoma Progression via Targeting miR-492 and miR-494-3p through Wnt/ß-catenin Signaling Pathway.

Purpose: Retinoblastoma (RB) is a frequent intraocular malignancy in children. Circular RNA (circRNA) plays an essential role in regulating the occurrence and development of tumors. This study aimed at investigating the function and molecular basis of hsa_circ_0093996 (circTET1) in RB.Methods: The expression of circTET1, miR-492 and miR-494-3p was examined using quantitative real-time polymerase chain reaction. Cell proliferation, cycle arrest, apoptosis, migration and invasion of RB cells were detected using Cell Counting Kit-8 (CCK-8), colony formation assay, flow cytometry, scratch assay and transwell analysis, respectively. The levels of matrix metalloproteinase (MMP) 2, MMP9 and Wnt/ß-catenin pathway-related proteins were measured via western blot assay. The association between circTET1 and miR-492/miR-494-3p was validated via dual-luciferase reporter assay and RNA pull-down assay. Xenograft assay was employed to analyze tumor growth in vivo.Results: CircTET1 level was reduced, while miR-492 and miR-494-3p levels were increased in RB tissues and cells. Overexpression of circTET1 inhibited proliferation, migration and invasion, and promoted apoptosis and cell cycle arrest in Y79 and WERI-Rb1 cells. Moreover, circTET1 impeded RB cell progression by sponging miR-492/miR-494-3p. Also, up-regulation of circTET1 restrained Wnt/ß-catenin pathway via regulating miR-492 and miR-494-3p. Furthermore, circTET1 suppressed tumor growth in xenograft models.Conclusion: CircTET1 inhibited RB progression by sponging miR-492/miR-494-3p and inactivating the Wnt/ß-catenin pathway, which provided new insights for RB treatment.