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Characterization of ENZ-resistant cell lines

Clinical characteristics of prostate cancer patients with matched diagnostic (archival) castration-sensitive and metastatic castration-resistant tissue biopsies

Purpose:Although enzalutamide (ENZ) has been widely used to treat de novo or castration-resistant metastatic prostate cancer, resistance develops and disease progression is ultimately inevitable. There are currently no approved targeted drugs to specifically delay or overcome ENZ resistance.Experimental Design:We selected several ENZ-resistant cell lines that replicated clinical characteristics of the majority of patients with ENZ-resistant disease. A high-throughput pharmacologic screen was utilized to identify compounds with greater cytotoxic effect for ENZ-resistant cell lines, compared with parental ENZ-sensitive cells. We validated the potential hits in vitro and in vivo, and used knockdown and overexpression assays to study the dependencies in ENZ-resistant prostate cancer.Results:ABT199 (BCL-2 inhibitor) and IMD0354 (IKKB inhibitor) were identified as potent and selective inhibitors of cell viability in ENZ-resistant cell lines in vitro and in vivo which were further validated using loss-of-function assays of BCL-2 and IKKB. Notably, we observed that overexpression of BCL-2 and IKKB in ENZ-sensitive cell lines was sufficient for the emergence of ENZ resistance. In addition, we confirmed that BCL-2 or IKKB inhibitors suppressed the development of ENZ resistance in xenografts. However, validation of both BCL-2 and IKKB in matched castration-sensitive/resistant clinical samples showed that, concurrent with the development of ENZ/abiraterone resistance in patients, only the protein levels of IKKB were increased.Conclusions:Our findings identify BCL-2 and IKKB dependencies in clinically relevant ENZ-resistant prostate cancer cells in vitro and in vivo, but indicate that IKKB upregulation appears to have greater relevance to the progression of human castrate-resistant prostate cancer.

List of antibodies used for Western Blot analyses

Compound full list of two libraries

List of primers used for qPCR analyses

Purpose: Although enzalutamide (ENZ) has been widely used to treat de novo or castration-resistant metastatic prostate cancer, resistance develops and disease progression is ultimately inevitable. There are currently no approved targeted drugs to specifically delay or overcome ENZ resistance. Experimental Design: We selected several ENZ-resistant cell lines that replicated clinical characteristics of the majority of patients with ENZ-resistant disease. A high-throughput pharmacologic screen was utilized to identify compounds with greater cytotoxic effect for ENZ-resistant cell lines, compared with parental ENZ-sensitive cells. We validated the potential hits in vitro and in vivo, and used knockdown and overexpression assays to study the dependencies in ENZ-resistant prostate cancer. Results: ABT199 (BCL-2 inhibitor) and IMD0354 (IKKB inhibitor) were identified as potent and selective inhibitors of cell viability in ENZ-resistant cell lines in vitro and in vivo which were further validated using loss-of-function assays of BCL-2 and IKKB. Notably, we observed that overexpression of BCL-2 and IKKB in ENZ-sensitive cell lines was sufficient for the emergence of ENZ resistance. In addition, we confirmed that BCL-2 or IKKB inhibitors suppressed the development of ENZ resistance in xenografts. However, validation of both BCL-2 and IKKB in matched castration-sensitive/resistant clinical samples showed that, concurrent with the development of ENZ/abiraterone resistance in patients, only the protein levels of IKKB were increased. Conclusions: Our findings identify BCL-2 and IKKB dependencies in clinically relevant ENZ-resistant prostate cancer cells in vitro and in vivo, but indicate that IKKB upregulation appears to have greater relevance to the progression of human castrate-resistant prostate cancer.

Eukaryotic elongation factor 1 alpha 2 (eEF1A2) is a transforming gene product that is highly expressed in human tumors of the ovary, lung, and breast. eEF1A2 also stimulates actin remodeling, and the expression of this factor is sufficient to induce the formation of filopodia, long cellular processes composed of bundles of parallel actin filaments. Here, we find that eEF1A2 stimulates formation of filopodia by increasing the cellular abundance of cytosolic and plasma membrane-bound phosphatidylinositol-4,5 bisphosphate [PI(4,5)P(2)]. We have previously reported that the eEF1A2 protein binds and activates phosphatidylinositol-4 kinase III beta (PI4KIIIbeta), and we find that production of eEF1A2-dependent PI(4,5)P(2) and generation of filopodia require PI4KIIIbeta. Furthermore, PI4KIIIbeta is itself capable of activating both the production of PI(4,5)P(2) and the creation of filopodia. We propose a model for extrusion of filopodia in which eEF1A2 activates PI4KIIIbeta, and activated PI4KIIIbeta stimulates production of PI(4,5)P(2) and filopodia by increasing PI4P abundance. Our work suggests an important role for both eEF1A2 and PI4KIIIbeta in the control of PI(4,5)P(2) signaling and actin remodeling.

eEF1A2 (eukaryotic protein elongation factor 1 alpha 2) is a protein translation factor that is likely a human oncogene by virtue of its capacity to transform mammalian cells and its high expression in tumors of the ovary, breast and lung. Here, we show that expression of eEF1A2 is sufficient to stimulate the formation of filopodia in BT549 human breast cancer cells and non-transformed Rat2 cells. Filopodia formation in eEF1A2-expressing cells is dependent on the activity of phosphatidylinositol-3 kinase (PI3K), and the ROCK and Akt kinases. Furthermore, eEF1A2 expression is sufficient to activate Akt in a PI3K-dependent fashion and inactivation of eEF1A2 by short interfering RNA reduces Akt activity. Using breast cancer cell line BT 549, we show that eEF1A2 expression stimulates cell migration and invasion in a largely PI3K- and Akt-dependent manner. These results suggest that eEF1A2 regulates oncogenesis through Akt and PI3K-dependent cytoskeletal remodeling.

Macrophages are generated through the differentiation of monocytes in tissues and they have important functions in innate and adaptive immunity. In addition to their roles as phagocytes, macrophages can be further differentiated, in the presence of receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF), into osteoclasts (multinucleated giant cells that are responsible for bone resorption). In this work, we set out to characterize whether various inflammatory stimuli, known to induce macrophage polarization, can alter the type of multinucleated giant cell obtained from RANKL differentiation. Following a four-day differentiation protocol, along with lipopolysaccharide (LPS)/interferon gamma (IFNγ) as one stimulus, and interleukin-4 (IL-4) as the other, three types of multinucleated cells were generated. Using various microscopy techniques (bright field, epifluorescence and scanning electron), functional assays, and western blotting for osteoclast markers, we found that, as expected, RANKL treatment alone resulted in osteoclasts, whereas the addition of LPS/IFNγ to RANKL pre-treated macrophages generated Langhans-type giant cells, while IL-4 led to giant cells resembling foreign body giant cells with osteoclast-like characteristics. Finally, to gain insight into the modulation of osteoclastogenesis, we characterized the formation and morphology of RANKL and LPS/IFNγ-induced multinucleated giant cells.

Macrophages can suppress Chlamydia replication by targeting the bacteria to degradative organelles such as lysosomes. Chlamydia trachomatis is an obligate intracellular bacterium responsible for one of the most common sexually transmitted diseases. In epithelial cells, C. trachomatis resides in a modified membrane-bound vacuole known as an inclusion, which is isolated from the endocytic pathway. However, the maturation process of C. trachomatis within immune cells, such as macrophages, has not been studied extensively. Here, we demonstrated that RAW macrophages effectively suppressed C. trachomatis growth and prevented Golgi stack disruption, a hallmark defect in epithelial cells after C. trachomatis infection. Next, we systematically examined association between C. trachomatis and various endocytic pathway markers. Spinning disk confocal time-lapse studies revealed significant and rapid association between C. trachomatis with Rab7 and LAMP1, markers of late endosomes and lysosomes. Moreover, pretreatment with an inhibitor of lysosome acidification led to significant increases in C. trachomatis growth in macrophages. At later stages of infection, C. trachomatis associated with the autophagy marker LC3. TEM analysis confirmed that a significant portion of C. trachomatis resided within double-membrane-bound compartments, characteristic of autophagosomes. Together, these results suggest that macrophages can suppress C. trachomatis growth by targeting it rapidly to lysosomes; moreover, autophagy is activated at later stages of infection and targets significant numbers of the invading bacteria, which may enhance subsequent chlamydial antigen presentation.

As major effector cells of the innate immune response, macrophages must adeptly migrate from blood to infected tissues. Endothelial transmigration is accomplished by matrix metalloproteinase (MMP)-induced degradation of basement membrane and extracellular matrix components. The classical activation of macrophages with LPS and IFN-γ causes enhanced microtubule (MT) stabilization and secretion of MMPs. Macrophages up-regulate MMP-9 expression and secretion upon immunological challenge and require its activity for migration during the inflammatory response. However, the dynamics of MMP-9 production and intracellular distribution as well as the mechanisms responsible for its trafficking are unknown. Using immunofluorescent imaging, we localized intracellular MMP-9 to small Golgi-derived cytoplasmic vesicles that contained calreticulin and protein-disulfide isomerase in activated RAW 264.7 macrophages. We demonstrated vesicular organelles of MMP-9 aligned along stable subsets of MTs and showed that selective modulation of MT dynamics contributes to the enhanced trafficking of MMP-9 extracellularly. We found a Rab3D-dependent association of MMP-9 vesicles with the molecular motor kinesin, whose association with the MT network was greatly enhanced after macrophage activation. Finally, we implicated kinesin 5B and 3B isoforms in the effective trafficking of MMP-9 extracellularly.

Introduction: Enzalutamide is a recently approved potent androgen receptor antagonist for prostate cancer both in the pre- and post-docetaxel settings. Its action relies upon 3 defined mechanisms of action: 1) blocking testosterone binding to the androgen receptor (AR), 2) preventing nuclear translocation of AR, and 3) inhibiting AR-DNA binding. However, despite its utility in prolonged overall survival in both these settings, disease relapse is inevitable, suggesting that resistance to the drug is somehow being acquired and is of vital concern. Methods: Using a combination of high-throughput functional genomic (kinome-shRNA) and chemical genomic (pharmacological inhibitor) screens, along with various in vitro / in vivo assays (proliferation, apoptosis, necrosis, soft agar growth, etc.) we have identified the IkB kinase (IKK) / IkB / NFkB signaling axis as important for acquisition of, and continued resistance to, Enzalutamide. Results: Using a panel of Enzalutamide-resistant and -sensitive cell lines, we show that targeting this signaling axis will selectively kill Enzalutamide-resistant cells while not affecting the proliferation of sensitive cells. Moreover, our data shows that the pharmacological inhibitors and shRNAs are effective even without Enzalutamide administration, negating any potential drug-drug interaction issues. Finally, we have found that our cells are resistant to Enzalutamide in an AR-V7-independent manner, suggesting that NFkB signaling is acting in a novel manner to bring about the resistance phenotype. Conclusions: Through in vitro and in vivo experiments, we provide evidence that targeting activators of NFκB signalling can be a strategy to help patients with Enzalutamide-resistant prostate cancer. Note: This abstract was not presented at the meeting. Citation Format: Sujeeve Jeganathan, Amina Zoubeidi, Martin Gleave, Brad G. Wouters, Anthony M. Joshua. Using functional and chemical genomics to identify mechanisms of Enzalutamide resistance in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 732. doi:10.1158/1538-7445.AM2015-732

Eukaryotic protein translation elongation factor 1 alpha 2 (eEF1A2) is an oncogene that transforms mammalian cell lines and increases their tumorigenicity in nude mice. Increased expression of eEF1A2 occurs during the development of breast, ovarian, and lung cancer. Here, we report that eEF1A2 directly binds to and activates phosphatidylinositol 4-kinase III beta (PI4KIIIbeta), an enzyme that converts phosphatidylinositol to phosphatidylinositol 4-phosphate. Purified recombinant eEF1A2 increases PI4KIIIbeta lipid kinase activity in vitro, and expression of eEF1A2 in rat and human cells is sufficient to increase overall cellular phosphatidylinositol 4-kinase activity and intracellular phosphatidylinositol 4-phosphate abundance. siRNA-mediated reduction in eEF1A2 expression concomitantly reduces phosphatidylinositol 4-kinase activity. This identifies a physical and functional relationship between eEF1A2 and PI4KIIIbeta.

Breast cancer is the most common malignancy among North American women. The identification of factors that predict outcome is key to individualized disease management and to our understanding of breast oncogenesis. We have analyzed mRNA expression of protein elongation factor eEF1A2 in two independent breast tumor populations of size n = 345 and n = 88, respectively. We find that eEF1A2 mRNA is expressed at a low level in normal breast epithelium but is detectably expressed in approximately 50-60% of primary human breast tumors. We have derived an eEF1A2-specific antibody and measured eEF1A2 protein expression in a sample of 438 primary breast tumors annotated with 20-year survival data. We find that high levels of eEF1A2 protein are detected in 60% of primary breast tumors independent of HER-2 protein expression, tumor size, lymph node status, and estrogen receptor (ER) expression. Importantly, we find that high eEF1A2 is a significant predictor of outcome. Women whose tumor has high eEF1A2 protein expression have an increased probability of 20-year survival compared to those women whose tumor does not express substantial eEF1A2. In addition, eEF1A2 protein expression predicts increased survival probability in those breast cancer patients whose tumor is HER-2 negative or who have lymph node involvement.