Improving the cytotoxicity of immunotoxins by reducing the affinity of the antibody in acidic pH.

BACKGROUND: Immunotoxins are antibody-toxin conjugates that bind to surface antigens and exert effective cytotoxic activity after internalization into tumor cells. Immunotoxins exhibit effective cytotoxicity and have been approved by the FDA to treat multiple hematological malignancies, such as hairy cell leukemia and cutaneous T-cell lymphoma. However, most of the internalized immunotoxin is degraded in lysosomes, and only approximately 5% of free toxin escapes into the cytosol to exert cytotoxicity. Many studies have improved immunotoxins by engineering the toxin fragment to reduce immunogenicity or increase stability, but how the antibody fragment contributes to the activity of immunotoxins has not been well demonstrated. METHODS: In the current study, we used 32A9 and 42A1, two anti-GPC3 antibodies with similar antigen-binding capabilities and internalization rates, to construct scFv-mPE24 immunotoxins and evaluated their in vitro and in vivo antitumor activities. Next, the antigen-binding capacity, trafficking, intracellular protein stability and release of free toxin of 32A9 scFv-mPE24 and 42A1 scFv-mPE24 were compared to elucidate their different antitumor activities. Furthermore, we used a lysosome inhibitor to evaluate the degradation behavior of 32A9 scFv-mPE24 and 42A1 scFv-mPE24. Finally, the antigen-binding patterns of 32A9 and 42A1 were compared under neutral and acidic pH conditions. RESULTS: Although 32A9 and 42A1 had similar antigen binding capacities and internalization rates, 32A9 scFv-mPE24 had superior antitumor activity compared to 42A1 scFv-mPE24. We found that 32A9 scFv-mPE24 exhibited faster degradation and drove efficient free toxin release compared to 42A1 scFv-mPE24. These phenomena were determined by the different degradation behaviors of 32A9 scFv-mPE24 and 42A1 scFv-mPE24 in lysosomes. Moreover, 32A9 was sensitive to the low-pH environment, which made the 32A9 conjugate easily lose antigen binding and undergo degradation in lysosomes, and the free toxin was then efficiently produced to exert cytotoxicity, whereas 42A1 was resistant to the acidic environment, which kept the 42A1 conjugate relatively stable in lysosomes and delayed the release of free toxin. CONCLUSIONS: These results showed that a low pH-sensitive antibody-based immunotoxin degraded faster in lysosomes, caused effective free toxin release, and led to improved cytotoxicity compared to an immunotoxin based on a normal antibody. Our findings suggested that a low pH-sensitive antibody might have an advantage in the design of immunotoxins and other lysosomal degradation-dependent antibody conjugate drugs.

The enhancer rare germline variation rs548071605 contributes to lung cancer development.

Rare germline variations contribute to the missing heritability of human complex diseases including cancers. Given their very low frequency, discovering and testing disease-causing rare germline variations remains challenging. The tag-single nucleotide polymorphism rs17728461 in 22q12.2 is highly associated with lung cancer risk. Here, we identified a functional rare germline variation rs548071605 (A>G) in a p65-responsive enhancer located within 22q12.2. The enhancer significantly promoted lung cancer cell proliferation in vitro and in a xenograft mouse model by upregulating the leukemia inhibitory factor (LIF) gene via the formation of a chromatin loop. Differential expression of LIF and its significant correlation with first progression survival time of patients further supported the lung cancer-driving effects of the 22q-Enh enhancer. Importantly, the rare variation was harbored in the p65 binding sequence and dramatically increased the enhancer activity by increasing responsiveness of the enhancer to p65 and B-cell lymphoma 3 protein, an oncoprotein that assisted the p65 binding. Our study revealed a regulatory rare germline variation with a potential lung cancer-driving role in the 22q12.2 risk region, providing intriguing clues for investigating the "missing heritability" of cancers, and also offered a useful experimental model for identifying causal rare variations.

Genetically Elevated LDL Associates with Lower Risk of Intracerebral Hemorrhage.

OBJECTIVE: Observational studies point to an inverse correlation between low-density lipoprotein (LDL) cholesterol levels and risk of intracerebral hemorrhage (ICH), but it remains unclear whether this association is causal. We tested the hypothesis that genetically elevated LDL is associated with reduced risk of ICH. METHODS: We constructed one polygenic risk score (PRS) per lipid trait (total cholesterol, LDL, high-density lipoprotein [HDL], and triglycerides) using independent genomewide significant single nucleotide polymorphisms (SNPs) for each trait. We used data from 316,428 individuals enrolled in the UK Biobank to estimate the effect of each PRS on its corresponding trait, and data from 1,286 ICH cases and 1,261 matched controls to estimate the effect of each PRS on ICH risk. We used these estimates to conduct Mendelian Randomization (MR) analyses. RESULTS: We identified 410, 339, 393, and 317 lipid-related SNPs for total cholesterol, LDL, HDL, and triglycerides, respectively. All four PRSs were strongly associated with their corresponding trait (all p < 1.00 × 10-100 ). While one SD increase in the PRSs for total cholesterol (odds ratio [OR] = 0.92; 95% confidence interval [CI] = 0.85-0.99; p = 0.03) and LDL cholesterol (OR = 0.88; 95% CI = 0.81-0.95; p = 0.002) were inversely associated with ICH risk, no significant associations were found for HDL and triglycerides (both p > 0.05). MR analyses indicated that 1mmol/L (38.67mg/dL) increase of genetically instrumented total and LDL cholesterol were associated with 23% (OR = 0.77; 95% CI = 0.65-0.98; p = 0.03) and 41% lower risks of ICH (OR = 0.59; 95% CI = 0.42-0.82; p = 0.002), respectively. INTERPRETATION: Genetically elevated LDL levels were associated with lower risk of ICH, providing support for a potential causal role of LDL cholesterol in ICH. ANN NEUROL 2020 ANN NEUROL 2020;88:56-66.

Shoot δ13 C values as an indicator of cumulative stress: The role of re-watering during drought and salinity stress.

RATIONALE: The carbon stable isotope composition (δ13 C value) of a plant can reflect prolonged drought and salinity, as different isotopic signals resulting from drought and salinity can be retained in plant tissue. Commonly, drought and salinity are interrupted by intermittent precipitation or irrigation. It remains unclear whether the δ13 C values reflect the cumulative duration of intermittent drought or salinity stress. METHODS: Drought (5% and 10% polyethylene glycol) and salinity (35 mM and 85 mM NaCl) were imposed on annual ryegrass consistently or cyclically; throughout the treatments, the stress duration for cyclic drought/salinity was half that of the corresponding prolonged stress treatment. The shoot δ13 C values were measured using isotope ratio mass spectrometry. RESULTS: Prolonged drought restrained growth and increased shoot δ13 C values relative to the control group. However, the shoot biomass was even lower under cyclic drought than under prolonged drought. Furthermore, the shoot δ13 C value under cyclic drought was close to that of the control group. The low NaCl concentration treatment actually enhanced shoot growth. The shoot δ13 C value varied with both duration and intensity of salinity across all groups. CONCLUSIONS: The shoot δ13 C value in annual ryegrass did indicate cumulative stress from cyclic low salinity, but not that from cyclic drought, in a manner that was mediated by the effect of re-watering on the mass and allocation of the photosynthates produced during stress.

Encapsulation of acetylshikonin by polyamidoamine dendrimers for preparing prominent nanoparticles.

Acetylshikonin (AS) has demonstrated antitumor potential. However, the development of therapeutic applications utilizing AS is inhibited by its poor solubility in water. In the present work, polyamidoamine (PAMAM) dendrimers and their PEGylated derivatives were employed to increase the solubility of AS. A distinct color transition was observed during the encapsulation of AS suggesting strong intermolecular forces between PAMAM and AS. Ultraviolet-visible, high-performance liquid chromatography, and (1)H NMR were used to verify the interaction between PAMAM and AS. The maximum amount of combined AS to each PAMAM molecule was determined. The cytotoxicity of AS nanoparticles was evaluated against leukemia (K562) and breast cancer (SK-BR-3) cell lines; the AS nanoparticles were shown to effectively inhibit tumor cells.

SATB1 mediated tumor colonization and ß-catenin nuclear localization are associated with colorectal cancer progression.

Colorectal cancer (CRC) is a malignancy with high incidence and poor prognosis. It is urgent to identify valuable biomarkers for early diagnosis and potent therapeutic targets. It has been reported that SATB1 is associated with the malignant progression in CRC. To explore the role of SATB1 in CRC progression and the underlying mechanism, we evaluated the expression of SATB1 in the paired CRC tissues with immunohistochemistry. The results showed that the expression of SATB1 in lymph node metastasis was higher than that in primary lesion, and that in distant organ metastasis was higher than that in primary lesion. The retrospective analysis showed that patients with high expression of SATB1 had a significantly worse prognosis than those with negative and moderate expression. In vitro experiments that employing SATB1 over-expressing and depleted CRC cell lines confirmed that SATB1 contributes to cell proliferation and colonization, while inhibiting cell motility. Furthermore, the tissue immunofluorescence assay, Co-IP and Western blot were conducted to reveal that SATB1 induced translocation of ß-catenin and formed a protein complex with it in the nuclei. In conclusion, SATB1 mediated tumor colonization and ß-catenin nuclear localization are associated with the malignant progression and poor prognosis of CRC.

Molecular characteristics and systemic treatment options of liposarcoma: A systematic review.

Liposarcoma (LPS) is a rare soft tissue sarcoma that develops from the differentiation of fat cells, typically occurring in the lower extremities and retroperitoneal space. Depending on its histological morphology and molecular changes, LPS can be divided into various subtypes, each exhibiting distinct biological behaviors. During treatment, especially for LPS arising in the retroperitoneum, the extent and quality of the initial surgery are critically important. Treatment strategies must be tailored to the specific type of LPS. Over the past few decades, the treatment of LPS has undergone numerous advancements, with new therapeutic approaches such as targeted drugs and immunotherapies continually emerging. This paper reviews the biological characteristics, molecular alterations, as well as surgical and pharmacological treatments of various LPS subtypes, with the aim of enhancing clinicians' understanding and emphasizing the importance of individualized precision therapy. With a deeper understanding of the biological characteristics and molecular alterations of LPS, future treatment trends are likely to focus more on developing personalized treatment plans to better address the various types of LPS.

The BCL2 gene is regulated by a special AT-rich sequence binding protein 1-mediated long range chromosomal interaction between the promoter and the distal element located within the 3'-UTR.

The 279-bp major breakpoint region (mbr) within the 3'-untranslated region (3'-UTR) of the BCL2 gene is a binding site of special AT-rich sequence binding protein 1 (SATB1) that is well known to participate in the long-range regulation of gene transcription. Our previous studies have revealed that the mbr could regulate BCL2 transcription over a 200-kb distance and this regulatory function was closely related to SATB1. This study is to explore the underlying mechanism and its relevance to cellular apoptosis. With chromosome conformation capture (3C) and chromatin immunoprecipitation (ChIP) assays we demonstrated that the mbr could physically interact with BCL2 promoter through SATB1-mediated chromatin looping, which was required for epigenetic modifications of the promoter, CREB accessibility and high expression of the BCL2 gene. During early apoptosis, SATB1 was a key regulator of BCL2 expression. Inhibition of SATB1 cleavage by treatment of cells with a caspase-6 inhibitor or overexpression of mutant SATB1 that was resistant to caspase-6, inhibited disassembly of the SATB1-mediated chromatin loop and restored the BCL2 mRNA level in Jurkat cells. These data revealed a novel mechanism of BCL2 regulation and mechanistically link SATB1-mediated long-range interaction with the regulation of a gene controlling apoptosis pathway for the first time.

Galectin-14 promotes hepatocellular carcinoma tumor growth via enhancing heparan sulfate proteoglycan modification.

Hepatocellular carcinoma (HCC) is a highly heterogeneous malignancy and lacks effective treatment. Bulk-sequencing of different gene transcripts by comparing HCC tissues and adjacent normal tissues provides some clues for investigating the mechanisms or identifying potential targets for tumor progression. However, genes that are exclusively expressed in a subpopulation of HCC may not be enriched or detected through such a screening. In the current study, we performed a single cell-clone-based screening and identified galectin-14 as an essential molecule in the regulation of tumor growth. The aberrant expression of galectin-14 was significantly associated with a poor overall survival of liver cancer patients with database analysis. Knocking down galectin-14 inhibited the proliferation of tumor growth, whereas overexpressing galectin-14 promoted tumor growth in vivo. Non-targeted metabolomics analysis indicated that knocking down galectin-14 decreased glycometabolism; specifically that glycoside synthesis was significantly changed. Further study found that galectin-14 promoted the expression of cell surface heparan sulfate proteoglycans (HSPGs) that functioned as co-receptors, thereby increasing the responsiveness of HCC cells to growth factors, such as epidermal growth factor and transforming growth factor-alpha. In conclusion, the current study identifies a novel HCC-specific molecule galectin-14, which increases the expression of cell surface HSPGs and the uptake of growth factors to promote HCC cell proliferation.

Improving antibody affinity through in vitro mutagenesis in complementarity determining regions.

High-affinity antibodies are widely used in diagnostics and for the treatment of human diseases. However, most antibodies are isolated from semi-synthetic libraries by phage display and do not possess in vivo affinity maturation, which is triggered by antigen immunization. It is therefore necessary to engineer the affinity of these antibodies by way of in vitro assaying. In this study, we optimized the affinity of two human monoclonal antibodies which were isolated by phage display in a previous related study. For the 42A1 antibody, which targets the liver cancer antigen glypican-3, the variant T57H in the second complementarity-determining region of the heavy chain (CDR-H2) exhibited a 2.6-fold improvement in affinity, as well as enhanced cell-binding activity. For the I4A3 antibody to severe acute respiratory syndrome coronavirus 2, beneficial single mutations in CDR-H2 and CDR-H3 were randomly combined to select the best synergistic mutations. Among these, the mutation S53P-S98T improved binding affinity (about 3.7 fold) and the neutralizing activity (about 12 fold) compared to the parent antibody. Taken together, single mutations of key residues in antibody CDRs were enough to increase binding affinity with improved antibody functions. The mutagenic combination of key residues in different CDRs creates additive enhancements. Therefore, this study provides a safe and effective in vitro strategy for optimizing antibody affinity.

Shed antigen-induced blocking effect on CAR-T cells targeting Glypican-3 in Hepatocellular Carcinoma.

BACKGROUND: Glypican-3 (GPC3), a cell surface glycoprotein that is pathologically highly expressed in hepatocellular carcinoma (HCC), is an attractive target for immunotherapies, including chimeric antigen receptor (CAR) T cells. The serum GPC3 is frequently elevated in HCC patients due to the shedding effect of cell surface GPC3. The shed GPC3 (sGPC3) is reported to block the function of cell-surface GPC3 as a negative regulator. Therefore, it would be worth investigating the potential influence of antigen shedding in anti-GPC3 CAR-T therapy for HCC. METHODS: In this study, we constructed two types of CAR-T cells targeting distinct epitopes of GPC3 to examine how sGPC3 influences the activation and cytotoxicity of CAR-T cells in vitro and in vivo by introducing sGPC3 positive patient serum or recombinant sGPC3 proteins into HCC cells or by using sGPC3-overexpressing HCC cell lines. RESULTS: Both humanized YP7 CAR-T cells and 32A9 CAR-T cells showed GPC3-specific antitumor functions in vitro and in vivo. The existence of sGPC3 significantly inhibited the release of cytokines and the cytotoxicity of anti-GPC3 CAR-T cells in vitro. In animal models, mice carrying Hep3B xenograft tumors expressing sGPC3 exhibited a worse response to the treatment with CAR-T cells under both a low and high tumor burden. sGPC3 bound to CAR-T cells but failed to induce the effective activation of CAR-T cells. Therefore, sGPC3 acted as dominant negative regulators when competed with cell surface GPC3 to bind anti-GPC3 CAR-T cells, leading to an inhibitory effect on CAR-T cells in HCC. CONCLUSIONS: We provide a proof-of-concept study demonstrating that GPC3 shedding might cause worse response to CAR-T cell treatment by competing with cell surface GPC3 for CAR-T cell binding, which revealed a new mechanism of tumor immune escape in HCC, providing a novel biomarker for patient enrolment in future clinical trials and/or treatments with GPC3-targeted CAR-T cells.

Poly (ADP-ribose) polymerase-1 binds to BCL2 major breakpoint region and regulates BCL2 expression.

BCL2, originally identified as a proto-oncogene in B-cell lymphoma, is a key regulator of apoptosis. Although it is more than 200 kb in length, at least 70% of the t(14;18) translocation in follicular lymphomas occurs at the BCL2 major breakpoint region (mbr), located in the 3'-untranslated region (3'-UTR). We have previously found that the mbr is a regulatory element which positively regulates BCL2 expression and this regulatory function was closely associated with SATB1, which binds to a 37 bp mbr (37 mbr) in the 3'-end of the mbr directly. However, the precise molecular mechanisms by which the mbr regulates gene expression are not fully understood. In this study, we purified Poly(ADP-ribose) polymerase-1 (PARP-1) from the DNA-protein complexes formed by 37 mbr in Jurkat cells and demonstrated that PARP-1 participates in the 37 mbr-protein complex's formation in vitro and in vivo. Functional analysis showed that overexpression of PARP-1 decreases 37 mbr regulatory function and BCL2 expression. Conversely, knockdown of PARP-1 with RNAi increases BCL2 expression. Taken together, the present findings indicate that PARP-1 is a component of BCL2 37 mbr-protein complexes, and PARP-1 is involved in the regulation of BCL2 expression. These findings are helpful in understanding the regulatory mechanisms of BCL2 expression.

Growth and carbohydrate dynamic of perennial ryegrass seedlings during PEG-simulated drought and subsequent recovery.

Due to the increasing occurrence of drought events, drought recovery has become equally important as drought resistance for long-term growth and survival of plants. However, information regarding the mechanism that controls growth recovery of herbaceous perennials is not available. In this study, perennial ryegrass (Lolium perenne) was rewatered after eight-day exposure to three drought intensities simulated by polyethylene glycol-6000. The growth, nonstructural carbohydrates (NSC, i.e. sucrose, glucose, fructose and starch), shoot δ13C, and activities of enzymes for sucrose conversion were monitored for 24 days after rewatering, allowing investigation of the dynamic of NSCs and its relation with growth in the recovery phase. In response to drought, growth and NSC content decreased mainly in shoot rather than root, and the total dry matter was negatively correlated to shoot δ13C. After rewatering, the growth of drought-treated groups still lagged behind that of control (CK) group for more than 16 days, but it was no longer correlated to shoot δ13C, suggesting that the limited growth is caused by non-stomatal factors related to photosynthesis. On day 24 after rewatering, the final growth of drought-treated groups caught up or even exceeded that of CK group, and was accompanied by higher dry weight root to shoot ratio (R/S) and root NSC content, which may facilitate water and nutrient acquisition and emergency of new tillers, respectively. During drought and subsequent recovery, the variation of R/S and root NSC content mainly attributed to root acid invertase rather than leaf sucrose phosphate synthase activity.

Effects of Wilting and Dew on the Water Isotope Composition of Detached Grass in Temperate Grassland.

Understanding the water isotopes in feed products derived from grass is fundamental for tracing domestic animal products. Grass silage water was reported to have fewer heavy isotopes than fresh grass, but it is still unknown whether dew formation (either dewfall or dewrise), exchange with soil water, or other processes override the expected enrichment of heavy isotopes due to wilting. The isotopic variations of water (δ2H, δ18O) in fresh grass and cut grass during wilting on soil and on plastic were compared in this study. Drying enriched heavier isotopes, but this was overridden by three processes that finally caused low δ2H and δ18O values: (i) the adsorption of humidity from the surroundings, (ii) the exchange with humidity, and (iii) the depletion of heavy water isotopes close to organic surfaces, called the surface effect, which was the most dominant effect at the end of drying when the water content became low.

The inherited variations of a p53-responsive enhancer in 13q12.12 confer lung cancer risk by attenuating TNFRSF19 expression.

BACKGROUND: Inherited factors contribute to lung cancer risk, but the mechanism is not well understood. Defining the biological consequence of GWAS hits in cancers is a promising strategy to elucidate the inherited mechanisms of cancers. The tag-SNP rs753955 (A>G) in 13q12.12 is highly associated with lung cancer risk in the Chinese population. Here, we systematically investigate the biological significance and the underlying mechanism behind 13q12.12 risk locus in vitro and in vivo. RESULTS: We characterize a novel p53-responsive enhancer with lung tissue cell specificity in a 49-kb high linkage disequilibrium block of rs753955. This enhancer harbors 3 highly linked common inherited variations (rs17336602, rs4770489, and rs34354770) and six p53 binding sequences either close to or located between the variations. The enhancer effectively protects normal lung cell lines against pulmonary carcinogen NNK-induced DNA damages and malignant transformation by upregulating TNFRSF19 through chromatin looping. These variations significantly weaken the enhancer activity by affecting its p53 response, especially when cells are exposed to NNK. The effect of the mutant enhancer alleles on TNFRSF19 target gene in vivo is supported by expression quantitative trait loci analysis of 117 Chinese NSCLC samples and GTEx data. Differentiated expression of TNFRSF19 and its statistical significant correlation with tumor TNM staging and patient survival indicate a suppressor role of TNFRSF19 in lung cancer. CONCLUSION: This study provides evidence of how the inherited variations in 13q12.12 contribute to lung cancer risk, highlighting the protective roles of the p53-responsive enhancer-mediated TNFRSF19 activation in lung cells under carcinogen stress.

Impact of anticancer drugs price cut on physician's prescription choices on first-line chemotherapy regimens and health expenditure for advanced non-small cell lung cancer in China.

BACKGROUND: Increases in insurance coverage and price cut of drugs are two important measures to make health care more accessible and affordable. As far as we know, this was the first study to explore the impact of anticancer drug price cut on health expenses and oncologist's prescription decisions in China. METHODS: The 511 non-small cell lung cancer (NSCLC) patients were recruited from Qilu Affiliated Hospital of Shandong University from January 1, 2003 to December 31, 2010. We categorized the patients into five groups based on China's fifth population census in 2000, including administrative group, workers and services group, peasants group, professionals group and others group. All statistical analyses were performed using SPSS (version 16.0), all statistic tests were two-tailed and P value ≤0.05 was considered significant. RESULTS: As for the first-line chemotherapy regimens prescribed during the study, 27.6% patients received vinorelbine + cisplatin (NP), 31.5% and 30.9% patients had gemcitabine + cisplatin (GC) and docetaxel + cisplatin (DC), respectively, while only 4.3% patients received paclitaxel + cisplatin or carboplatin (TP). Before price policy implementation, NP was the most popularly used regimen (44.6%). By contrast, doctors' prescription choices changed significantly after drug price cut, GC took first place (42.0%). GC became the most expensive regimen (4,431.40 RMB per cycle, about 665.15 dollars per cycle), while NP cost the least (1,974.48 RMB per cycle, about 296.37 dollars per cycle) after price cut. No significant reduction could be seen for both the pharmaceutical spending and total expense per inpatient episode after drug price adjustment. One interesting phenomena was that doctors relied less on patient's sex, age, histology to make their decisions, by contrast, more on patient's occupation and health insurance type. And, the total drug cost was closely related to patient occupation and health insurance type. CONCLUSIONS: The introduction of anticancer drug price control policy was found to be ineffective on the containment of hospital drug expenditures in one cancer center in China.

ERα propelled aberrant global DNA hypermethylation by activating the DNMT1 gene to enhance anticancer drug resistance in human breast cancer cells.

Drug-induced aberrant DNA methylation is the first identified epigenetic marker involved in chemotherapy resistance. Understanding how the aberrant DNA methylation is acquired would impact cancer treatment in theory and practice. In this study we systematically investigated whether and how ERα propelled aberrant global DNA hypermethylation in the context of breast cancer drug resistance. Our data demonstrated that anticancer drug paclitaxel (PTX) augmented ERα binding to the DNMT1 and DNMT3b promoters to activate DNMT1 and DNMT3b genes, enhancing the PTX resistance of breast cancer cells. In support of these observations, estrogen enhanced multi-drug resistance of breast cancer cells by up-regulation of DNMT1 and DNMT3b genes. Nevertheless, the aberrant global DNA hypermethylation was dominantly induced by ERα-activated-DNMT1, since DNMT1 over-expression significantly increased global DNA methylation and DNMT1 knockdown reversed the ERα-induced global DNA methylation. Altering DNMT3b expression had no detectable effect on global DNA methylation. Consistently, the expression level of DNMT1 was positively correlated with ERα in 78 breast cancer tissue samples shown by our immunohistochemistry (IHC) analysis and negatively correlated with relapse-free survival (RFS) and distance metastasis-free survival (DMFS) of ERα-positive breast cancer patients. This study provides a new perspective for understanding the mechanism underlying drug-resistance-facilitating aberrant DNA methylation in breast cancer and other estrogen dependent tumors.

LncRNA H19 confers chemoresistance in ERα-positive breast cancer through epigenetic silencing of the pro-apoptotic gene BIK.

Breast cancer is a common malignancy in women. Acquisition of drug resistance is one of the main obstacles encountered in breast cancer therapy. Long non-coding RNA (lncRNA) has been demonstrated to play vital roles in both development and tumorigenesis. However, the relationship between lncRNAs and the development of chemoresistance is not well established. In the present study, the high expression of lncRNA H19 was identified as a powerful factor associated with paclitaxel (PTX) resistance in ERα-positive breast cancer cells, but not in ERα-negative breast cancer cells. LncRNA H19 attenuated cell apoptosis in response to PTX treatment by inhibiting transcription of pro-apoptotic genes BIK and NOXA. H19 was further confirmed to suppress the promoter activity of BIK by recruiting EZH2 and by trimethylating the histone H3 at lysine 27. Interestingly, our data showed that lncRNA H19 was one of the downstream target molecules of ERα. Altered ERα expression may therefore change H19 levels to modulate the apoptosis response to chemotherapy in breast cancer cells. Our data suggest that the ERα-H19-BIK signaling axis plays an important role in promoting chemoresistance.

SATB1 Mediates Long-Range Chromatin Interactions: A Dual Regulator of Anti-Apoptotic BCL2 and Pro-Apoptotic NOXA Genes.

Aberrant expression of special AT-rich binding protein 1 (SATB1), a global genomic organizer, has been associated with various cancers, which raises the question of how higher-order chromatin structure contributes to carcinogenesis. Disruption of apoptosis is one of the hallmarks of cancer. We previously demonstrated that SATB1 mediated specific long-range chromosomal interactions between the mbr enhancer located within 3'-UTR of the BCL2 gene and the promoter to regulate BCL2 expression during early apoptosis. In the present study, we used chromosome conformation capture (3C) assays and molecular analyses to further investigate the function of the SATB1-mediated higher-order chromatin structure in co-regulation of the anti-apoptotic BCL2 gene and the pro-apoptotic NOXA gene located 3.4Mb downstream on Chromosome 18. We demonstrated that the mbr enhancer spatially juxtaposed the promoters of BCL2 and NOXA genes through SATB1-mediated chromatin-loop in Jurkat cells. Decreased SATB1 levels switched the mbr-BCL2 loop to mbr-NOXA loop, and thus changed expression of these two genes. The SATB1-mediated dynamic switch of the chromatin loop structures was essential for the cooperative expression of the BCL2 and NOXA genes in apoptosis. Notably, the role of SATB1 was specific, since inhibition of SATB1 degradation by caspase-6 inhibitor or caspase-6-resistant SATB1 mutant reversed expression of BCL-2 and NOXA in response to apoptotic stimulation. This study reveals the critical role of SATB1-organized higher-order chromatin structure in regulating the dynamic equilibrium of apoptosis-controlling genes with antagonistic functions and suggests that aberrant SATB1 expression might contribute to cancer development by disrupting the co-regulated genes in apoptosis pathways.

A novel SATB1 binding site in the BCL2 promoter region possesses transcriptional regulatory function.

BCL2 is a key regulator of apoptosis. Our previous work has demonstrated that special AT-rich sequence-binding protein 1 (SATB1) is positively correlated with BCL2 expression. In the present study, we report a new SATB1 binding site located between P1 and P2 promoters of the BCL2 gene. The candidate SATB1 binding sequence predicted by bioinformatic analysis was investigated in vitro and in vivo by electrophoretic gel mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP). One 25-bp sequence, named SB1, was confirmed to be SATB1 binding site. The regulatory function of SB1 and its relevance to SATB1 were further examed with dual-luciferase reporter assay system in Jurkat cells. We found that SB1 could negatively regulate reporter gene activity. Mutation of SATB1 binding site further repressed the activity. Knockdown of SATB1 also enhanced this negative effect of SB1. Our data indicate that the SB1 sequence possesses negative transcriptional regulatory function and this function can be antagonized by SATB1.