Knockdown of dual specificity phosphatase 4 enhances the chemosensitivity of MCF-7 and MCF-7/ADR breast cancer cells to doxorubicin

BackgroundBreast cancer is the major cause of cancer-related deaths in females world-wide. Doxorubicin-based therapy has limited efficacy in breast cancer due to drug resistance, which has been shown to be associated with the epithelial-to-mesenchymal transition (EMT). However, the molecular mechanisms linking the EMT and drug resistance in breast cancer cells remain unclear. Dual specificity phosphatase 4 (DUSP4), a member of the dual specificity phosphatase family, is associated with cellular proliferation and differentiation; however, its role in breast cancer progression is controversial.MethodsWe used cell viability assays, Western blotting and immunofluorescent staining, combined with siRNA interference, to evaluate chemoresistance and the EMT in MCF-7 and adriamycin-resistant MCF-7/ADR breast cancer cells, and investigate the underlying mechanisms.ResultsKnockdown of DUSP4 significantly increased the chemosensitivity of MCF-7 and MCF-7/ADR breast cancer cells to doxorubicin, and MCF-7/ADR cells which expressed high levels of DUSP4 had a mesenchymal phenotype. Furthermore, knockdown of DUSP4 reversed the EMT in MCF-7/ADR cells, as demonstrated by upregulation of epithelial biomarkers and downregulation of mesenchymal biomarkers, and also increased the chemosensitivity of MCF-7/ADR cells to doxorubicin.ConclusionsDUSP4 might represent a potential drug target for inhibiting drug resistance and regulating the process of the EMT during the treatment of breast cancer.     

Phase I Trial: Cirmtuzumab Inhibits ROR1 Signaling and Stemness Signatures in Patients with Chronic Lymphocytic Leukemia

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Production of functional human CuZn-SOD and EC-SOD in bitransgenic cloned goat milk

Human copper/zinc superoxide dismutase (CuZn-SOD) and extracellular superoxide dismutase (EC-SOD) are two superoxide dismutases that scavenge reactive oxygen species (ROS). Their biological role of eliminating oxidative stress caused by excessive ROS levels in living organisms has been utilized in medical treatment, preventing skin photoaging and food preservation. In this study, we employed two sequences that encode human CuZn-SOD and EC-SOD, along with goat beta-casein 5′ and 3′ regulatory elements, to construct mammary gland-specific expression vectors. Bitransgenic goats were generated using somatic cell nuclear transfer (SCNT), which employed co-transfection to generate bitransgenic goat fetal fibroblast cells as donor cells, and the expression of human CuZn-SOD and EC-SOD and their biological activities were assayed in the milk. PCR and Southern blot analysis confirmed that the cloned goat harbors both hCuZn-SOD and hEC-SOD transgenes. rhCuZn-SOD and rhEC-SOD were expressed in the mammary glands of bitransgenic goat, as determined by western blotting. The expression levels were 100.14?±?5.09 mg/L for rhCuZn-SOD and 279.10?±?5.38 mg/L for rhEC-SOD, as determined using ELISA. A total superoxide dismutase assay with WST-8 indicates that the biological activity of rhCuZn-SOD and rhEC-SOD in goat milk is 1451?±?136 U/mL. The results indicate that two expression vectors can simultaneously transfect goat fetal fibroblast cells as donor cells to produce transgenic goats by SCNT, and the CuZn-SOD and EC-SOD proteins secreted in the mammary glands showed biological activity. The present study thus describes an initial step in the production of recombinant human SODs that may potentially be used for therapeutic purposes.     

A Pan-BCL2 Inhibitor Renders Bone-Marrow-Resident Human Leukemia Stem Cells Sensitive to Tyrosine Kinase Inhibition

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Highlights? Splice-isoform switching favors prosurvival BCL2 family expression in human BC ? BC LSCs are quiescent and TKI-resistant in the marrow niche ? Sabutoclax, a pan-BCL2 inhibitor, enhances TKI sensitivity of bone marrow BC LSCs     

Kinetics of the processes,plasma parameters,and output characteristics of a UV emitter operating on XeI molecules and iodine molecules and atoms

A kinetic model of the processes occurring in the plasma of a high-power low-pressure gas-discharge lamp is presented, and the output characteristics of the lamp are described. The lamp is excited by a longitudinal glow discharge and emits the I₂(D′-A′) 342-nm and XeI(B-X) 253-nm bands and the 206.2-nm spectral line of atomic iodine. When the emitter operates in a sealed-off mode on the p(He): p(Xe): p(I₂) = 400: 120: (100–200) Pa mixture, the fractions of the UV radiation power of iodine atoms, exciplex molecules of xenon iodide, and iodine molecules comprise 55, 10, and 35%, respectively. At the optimal partial pressure, the maximum total radiation power of the lamp reaches 37 W, the energy efficiency being about 15%.     

A family of Bayes multiple testing procedures

Under the model of independent test statistics, we propose atwo-parameter family of Bayes multiple testing procedures. Thetwo parameters can be viewed as tuning parameters. Using theBenjamini–Hochberg step-up procedure for controlling falsediscovery rate as a baseline for conservativeness, we choosethe tuning parameters to compromise between the operating characteristicsof that procedure and a less conservative procedure that focuseson alternatives that a priori might be considered likely ormeaningful. The Bayes procedures do not have the theoreticaland practical shortcomings of the popular stepwise procedures.In terms of the number of mistakes, simulations for two examplesindicate that over a large segment of the parameter space, theBayes procedure is preferable to the step-up procedure. Anotherdesirable feature of the procedures is that they are computationallyfeasible for any number of hypotheses.     

Identification,purification, and pharmacological activity analysis of Desmodus rotundus salivary plasminogen activator alpha1 (DSPAα1) expressed in transgenic rabbit mammary glands

Desmodus rotundus plasminogen activator alpha 1(DSPAα1) is a thrombolytic protein with advantages, such as a long half-life, high accuracy and specificity for thrombolysis, wide therapeutic window, and no neurotoxicity. To date, DSPAα1 has only been expressed in the Chinese hamster ovary, insect cells, transgenic tobacco plants, and Pichia pastoris. To the best of our knowledge, we are the first to report the expression of DSPAα1 in transgenic rabbit mammary glands, extract the product, and analyze its pharmacology activity. An efficient mammary gland-specific expression vector pCL25/DSPAα1 was transferred to prokaryotic zygotes in rabbits by microinjection to generate six DSPAα1 transgenic rabbits. The recombinant DSPAα1 (rDSPAα1) expression in transgenic rabbit milk was 1.19?±?0.26 mg/mL. The rDSPAα1 purification protocol included pretreatment, ammonium sulfate precipitation, benzamidine affinity chromatography, cation exchange chromatography, and Cibacron blue affinity chromatography; approximately 98% purity was achieved using gel electrophoresis. According to sequencing results, the primary structure of rDSPAα1 was consistent with the theoretical design sequence, and its molecular weight was consistent with that of the natural protein. N-terminal sequencing results indicated rDSPAα1 to be a mature protein, as the goat signal peptide sequence of the expression vector was no longer detected. The fibrinolytic activity of rDSPAα1 was estimated to be 773,333 IU/mg. Fibrin-agarose plate assay and in vitro rat blood clot degradation assay showed that rDSPAα1 had strong thrombolytic activity. In conclusion, we report recombinant DSPAα1 with high thrombolytic activity expressed in transgenic rabbit mammary glands.

     

Mutational Profiling Can Establish Clonal or Independent Origin in Synchronous Bilateral Breast and Other Tumors

Background

Synchronous tumors can be independent primary tumors or a primary-metastatic (clonal) pair, which may have clinical implications. Mutational profiling of tumor DNA is increasingly common in the clinic. We investigated whether mutational profiling can distinguish independent from clonal tumors in breast and other cancers, using a carefully defined test based on the Clonal Likelihood Score (CLS = 100 x # shared high confidence (HC) mutations/ # total HC mutations).

Methods

Statistical properties of a formal test using the CLS were investigated. A high CLS is evidence in favor of clonality; the test is implemented as a one-sided binomial test of proportions. Test parameters were empirically determined using 16,422 independent breast tumor pairs and 15 primary-metastatic tumor pairs from 10 cancer types using The Cancer Genome Atlas.

Results

We validated performance of the test with its established parameters, using five published data sets comprising 15,758 known independent tumor pairs (maximum CLS = 4.1%, minimum p-value = 0.48) and 283 known tumor clonal pairs (minimum CLS 13%, maximum p-value <0.01), across renal cell, testicular, and colorectal cancer. The CLS test correctly classified all validation samples but one, which it appears may have been incorrectly classified in the published data. As proof-of-concept we then applied the CLS test to two new cases of invasive synchronous bilateral breast cancer at our institution, each with one hormone receptor positive (ER+/PR+/HER2-) lobular and one triple negative ductal carcinoma. High confidence mutations were identified by exome sequencing and results were validated using deep targeted sequencing. The first tumor pair had CLS of 81% (p-value < 10–15), supporting clonality. In the second pair, no common mutations of 184 variants were validated (p-value >0.99), supporting independence. A plausible molecular mechanism for the shift from hormone receptor positive to triple negative was identified in the clonal pair.

Conclusion

We have developed the statistical properties of a carefully defined Clonal Likelihood Score test from mutational profiling of tumor DNA. Under identified conditions, the test appears to reliably distinguish between synchronous tumors of clonal and of independent origin in several cancer types. This approach may have scientific and clinical utility.