Dale Corkery

Leukaemia xenotransplantation in zebrafish – chemotherapy response assay in vivo

As the molecular understanding of cancer pathogenesis has evolved, so has the need for innovative assays to identify novel targeted therapeutic agents. Characterization of specific tumour-drug interactions would enable the design of more personalized treatment with improved outcomes and reduced toxicity. The zebrafish has emerged as a robust animal model of human malignancy due to conserved genetics and cell biology (Amatruda & Patton, 2008; Payne & Look, 2009). Xenotransplantation of human cancer cells into zebrafish was initiated in 2005 using a melanoma xenograft (Lee et al, 2005). Subsequent studies determined that incubation at 35 C enabled normal growth of injected human cell lines without compromising zebrafish embryogenesis and established the yolk sac and 48 h post-fertilization (hpf) as the ideal anatomic location and developmental stage for injection (Haldi et al, 2006). At this stage, adaptive immune responses are not yet established in the embryo, permitting injection of human cells without requiring immunosuppression (Lam et al, 2004; Haldi et al, 2006; Nicoli et al, 2007). Proliferation of xenografted melanoma cells was documented using a haemocytometer (Haldi et al, 2006); while proliferation of xenografted leukaemia cells was determined by fluorescence and embryo demise (Pruvot et al, 2011). These reports provided a critical foundation, however further refinement and proof-of-principle studies are necessary for this approach to be practically applied to cancer drug discovery and clinically employed for therapeutic drug responses. To this end, we transplanted K562 and NB-4 human leukaemia cell lines into casper embryos, a zebrafish combinatorial pigment mutant (White et al, 2008), and developed a robust cell proliferation assay demonstrating in vivo targeted therapeutic inhibition of these leukaemias. Five million K562 or NB-4 cells were pelleted by centrifugation (5 min at 100 g), resuspended in phosphatebuffered saline (PBS) containing CM-DiI (Invitrogen, Burlington, ON, Canada) (5 lg/ml) and incubated for 5 min at 37 C and 20 min at 4 C. Cells were pelleted, washed twice with PBS, and re-suspended in 500 ll RPMI medium. Dechorinated 48-h casper embryos (adults maintained as described by Westerfield, 1995) were anaesthetized with tricaine and 25–50 labelled cells injected into the yolk sac (adapted from (Haldi et al, 2006)). Following 1 h recovery at 28 C, embryos were maintained at 35 C, and screened for fluorescence at the injection site. At 24 h postinjection (hpi), imatinib mesylate (IM), all-trans retinoic acid (ATRA), or vehicle (dimethyl sulfoxide, DMSO) were added to the water for 48 h. Proliferation of leukaemia cells was monitored by live-cell microscopy. Embryos (15–20) were dissociated at 24 and 72 hpi with or without drug, incubated in protease solution (0Æ25% trypsin, 1 mmol/l EDTA, 2Æ25 mg/ml collagenase (Sigma-Aldrich, Oakville, ON, Canada) in 1Æ2 ml sterile PBS) for 45 min at 35 C and homogenized. Cells were pelleted by centrifugation (400 g · 5 min) and resuspended in 0Æ9· PBS with 5% fetal bovine serum at 10 ll per embryo (Fig. 1). Droplets were imaged as a 5 · 4 mosaic (Axio Observer Z, Colibri LED light source, Axiocam Rev 3.0 CCD camera, Carl Zeiss, Wetzlar, Germany) (Fig. S1). Leukaemia cells were counted in four internal fields using a semi-automated macro (image j; NIH, Bethesda, MD, USA). Co-staining with 10 nmol/l DRAQ5 viable nuclear stain confirmed enumeration of intact leukaemia cells (Biostatus Ltd. Leicester, UK) (Fig. S2). Using four micrographs from five boluses repeated in triplicate, average cell counts per embryo were determined. The use of zebrafish in these experiments was approved by the Dalhousie University Committee on Laboratory Animals. Injected embryos tolerated the presence of human leukaemia cells for up to 7 days, during which time engrafted leukaemia cells proliferated and circulated within the embryonic vasculature (Fig. 2A; Movies S1 and S2). K562 cell numbers increased from 90 to 230 cells per embryo (2Æ6fold) and NB-4 cells from 50 to 170 per embryo (3Æ4-fold) after 48 h (Fig. 2B). To evaluate this xenotransplantation assay as a means of determining specific tumour-drug interactions in vivo, we exploited the known responses to targeted therapies of the molecular alterations in each leukaemia cell line. Specifically, we used IM to target BCR-ABL1 in engrafted K562 cells and ATRA to target PML-RARA in engrafted NB-4 cells. Toxicity curves were generated for both 48-h K562 xenotransplanted zebrafish embryos exposed to IM and NB-4 xenotransplanted embryos treated with ATRA (Fig. S3). We subsequently used 50% of the maximum tolerated dose (MTD) (20 lmol/l IM; 0Æ2 lmol/l ATRA). K562 xenotransplanted embryos exposed to IM or NB-4 xenotransplanted embryos exposed to ATRA for 48 h, displayed a significant decrease in leukaemia cells compared to vehicle-treated control embryos (P < 0Æ001) (Fig. 2C). Reciprocal experiments failed to show a significant inhibition in proliferation (Fig. 2C), confirming that the correspondence

Translational Activation of HIF1α by YB-1 Promotes Sarcoma Metastasis

Metastatic dissemination is the leading cause of death in cancer patients, which is particularly evident for high-risk sarcomas such as Ewing sarcoma, osteosarcoma, and rhabdomyosarcoma. Previous research identified a crucial role for YB-1 in the epithelial-to-mesenchymal transition (EMT) and metastasis of epithelial malignancies. Based on clinical data and two distinct animal models, we now report that YB-1 is also a major metastatic driver in high-risk sarcomas. Our data establish YB-1 as a critical regulator of hypoxia-inducible factor 1α (HIF1α) expression in sarcoma cells. YB-1 enhances HIF1α protein expression by directly binding to and activating translation of HIF1A messages. This leads to HIF1α-mediated sarcoma cell invasion and enhanced metastatic capacity in vivo, highlighting a translationally regulated YB-1-HIF1α axis in sarcoma metastasis.

Focused chemical genomics using zebrafish xenotransplantation as a pre-clinical therapeutic platform for T-cell acute lymphoblastic leukemia

Cancer therapeutics is evolving to precision medicine, with the goal of matching targeted compounds with molecular aberrations underlying a patient’s cancer. While murine models offer a pre-clinical tool, associated costs and time are not compatible with actionable patient-directed interventions. Using the paradigm of T-cell acute lymphoblastic leukemia, a high-risk disease with defined molecular underpinnings, we developed a zebrafish human cancer xenotransplantation model to inform therapeutic decisions. Using a focused chemical genomic approach, we demonstrate that xenografted cell lines harboring mutations in the NOTCH1 and PI3K/AKT pathways respond concordantly to their targeted therapies, patient-derived T-cell acute lymphoblastic leukemia can be successfully engrafted in zebrafish and specific drug responses can be quantitatively determined. Using this approach, we identified a mutation sensitive to γ-secretase inhibition in a xenograft from a child with T-cell acute lymphoblastic leukemia, confirmed by Sanger sequencing and validated as a gain-of-function NOTCH1 mutation. The zebrafish xenotransplantation platform provides a novel cost-effective means of tailoring leukemia therapy in real time.

Connecting the speckles: Splicing kinases and their role in tumorigenesis and treatment response

Alternative pre-mRNA splicing in higher eukaryotes enhances transcriptome complexity and proteome diversity. Its regulation is mediated by a complex RNA-protein network that is essential for the maintenance of cellular and tissue homeostasis. Disruptions to this regulatory network underlie a host of human diseases and contribute to cancer development and progression. The splicing kinases are an important family of pre-mRNA splicing regulators, , which includes the CDC-like kinases (CLKs), the SRSF protein kinases (SRPKs) and pre-mRNA splicing 4 kinase (PRP4K/PRPF4B). These splicing kinases regulate pre-mRNA splicing via phosphorylation of spliceosomal components and serine-arginine (SR) proteins, affecting both their nuclear localization within nuclear speckle domains as well as their nucleo-cytoplasmic shuttling. Here we summarize the emerging evidence that splicing kinases are dysregulated in cancer and play important roles in both tumorigenesis as well as therapeutic response to radiation and chemotherapy.

Synthesis and biological evaluation of prodigiosene conjugates of porphyrin, estrone and 4-hydroxytamoxifen

To generate the first series of prodigiosene conjugates, the tripyrrolic skeleton was appended to estrone, tamoxifen and porphyrin frameworks by way of ester linkers and various hydrocarbon chain lengths. The ability of the conjugates to inhibit various types of cancer cells was evaluated in vitro. The porphyrin conjugates did not exhibit significant activity. The estrone conjugates exhibited modest activity, for the most part. However, significantly greater growth inhibition activity against certain breast, colon, lung, leukemia, melanoma and prostate cell lines was noted. This unusual effect for this first generation model class of compound warrants further investigation and comparison to cases where estrogens are linked to prodigiosenes via connection points that do not feature in estrogen receptor binding. The 4-hydroxytamoxifen conjugates exhibit nanomolar range activity against the MCF-7 breast cancer cell line, paving the way to expand the scope and connectivity of prodigiosene-tamoxifen conjugates.

The translation initiation factor 3 subunit eIF3K interacts with PML and associates with PML nuclear bodies.

The promyelocytic leukemia protein (PML) is a tumor suppressor protein that regulates a variety of important cellular processes, including gene expression, DNA repair and cell fate decisions. Integral to its function is the ability of PML to form nuclear bodies (NBs) that serve as hubs for the interaction and modification of over 90 cellular proteins. There are seven canonical isoforms of PML, which encode diverse C-termini generated by alternative pre-mRNA splicing. Recruitment of specific cellular proteins to PML NBs is mediated by protein-protein interactions with individual PML isoforms. Using a yeast two-hybrid screen employing peptide sequences unique to PML isoform I (PML-I), we identified an interaction with the eukaryotic initiation factor 3 subunit K (eIF3K), and in the process identified a novel eIF3K isoform, which we term eIF3K-2. We further demonstrate that eIF3K and PML interact both in vitro via pull-down assays, as well as in vivo within human cells by co-immunoprecipitation and co-immunofluorescence. In addition, eIF3K isoform 2 (eIF3K-2) colocalizes to PML bodies, particularly those enriched in PML-I, while eIF3K isoform 1 associates poorly with PML NBs. Thus, we report eIF3K as the first known subunit of the eIF3 translation pre-initiation complex to interact directly with the PML protein, and provide data implicating alternative splicing of both PML and eIF3K as a possible regulatory mechanism for eIF3K localization at PML NBs.

PRP4K is a HER2-regulated modifier of taxane sensitivity

The taxanes are used alone or in combination with anthracyclines or platinum drugs to treat breast and ovarian cancer, respectively. Taxanes target microtubules in cancer cells and modifiers of taxane sensitivity have been identified in vitro, including drug efflux and mitotic checkpoint proteins. Human epidermal growth factor receptor 2 (HER2/ERBB2) gene amplification is associated with benefit from taxane therapy in breast cancer yet high HER2 expression also correlates with poor survival in both breast and ovarian cancer. The pre-mRNA splicing factor 4 kinase PRP4K (PRPF4B), which we identified as a component of the U5 snRNP also plays a role in regulating the spindle assembly checkpoint (SAC) in response to microtubule-targeting drugs. In this study, we found a positive correlation between PRP4K expression and HER2 status in breast and ovarian cancer patient tumors, which we determined was a direct result of PRP4K regulation by HER2 signaling. Knock-down of PRP4K expression reduced the sensitivity of breast and ovarian cancer cell lines to taxanes, and low PRP4K levels correlated with in vitro-derived and patient acquired taxane resistance in breast and ovarian cancer. Patients with high-grade serous ovarian cancer and high HER2 levels had poor overall survival; however, better survival in the low HER2 patient subgroup treated with platinum/taxane-based therapy correlated positively with PRP4K expression (HR = 0.37 [95% CI 0.15-0.88]; p = 0.03). Thus, PRP4K functions as a HER2-regulated modifier of taxane sensitivity that may have prognostic value as a marker of better overall survival in taxane-treated ovarian cancer patients.

ErbB2-dependent downregulation of a pro-apoptotic protein Perp is required for oncogenic transformation of breast epithelial cells

The ability of breast cancer cells to resist anoikis, apoptosis caused by detachment of the non-malignant epithelial cells from the extracellular matrix (ECM), is thought to be critical for breast tumor growth, invasion and metastasis. ErbB2, an oncoprotein that is often overproduced in breast tumors, can block breast cancer cell anoikis via mechanisms that are understood only in part. In an effort to understand them better we found that detachment of the non-malignant human breast epithelial cells from the ECM upregulates a protein Perp in these cells. Perp is a component of the desmosomes, multiprotein complexes involved in cell-to-cell adhesion. Perp can cause apoptosis via unknown mechanisms. We demonstrated that Perp upregulation by cell detachment is driven by detachment-induced loss of epidermal growth factor receptor (EGFR). We also found that Perp knockdown by RNA interference (RNAi) rescues detached cells from death which indicates that Perp contributes to their anoikis. We observed that ErbB2, when overexpressed in detached breast epithelial cells, causes Perp downregulation. Furthermore, ErbB2-directed RNAi or treatment with lapatinib, an ErbB2/EGFR small-molecule inhibitor used for breast cancer therapy, upregulated Perp in ErbB2-positive human breast and ovarian carcinoma cells. We established that ErbB2 downregulates Perp by activating an ErbB2 effector protein kinase Mek that blocks detachment-induced EGFR loss in a manner that requires the presence of a signaling protein Sprouty-2. Finally, we observed that restoration of the wild-type Perp levels in ErbB2-overproducing breast epithelial cells increases their anoikis susceptibility and blocks their clonogenicity in the absence of adhesion to the ECM. In summary, we have identified a novel mechanism of ErbB2-mediated mechanism of anoikis resistance of ErbB2-overproducing breast epithelial cells. This mechanism allows such cells to grow without adhesion to the ECM and is driven by ErbB2-induced activation of Mek, subsequent EGFR upregulation and further EGFR-dependent Perp loss.

Estrogen receptor alpha (ESR1)-signaling regulates the expression of the taxane-response biomarker PRP4K.

The pre-mRNA splicing factor 4 kinase PRP4K (PRPF4B), is an essential kinase that is a component of the U5 snRNP and functions in spliceosome assembly. We demonstrated that PRP4K is a novel biological marker for taxane response in ovarian cancer patients and reduced levels of PRP4K correlate with intrinsic and acquired taxane resistance in both breast and ovarian cancer. Breast cancer treatments are chosen based on hormone and growth factor receptor status, with HER2 (ERBB2) positive breast cancer patients receiving anti-HER2 agents and taxanes and estrogen receptor alpha (ESR1) positive (ER+) breast cancer patients receiving anti-estrogen therapies such as tamoxifen. Here we demonstrate that PRP4K is expressed in the normal mammary duct epithelial cells of the mouse, and that estrogen induces PRP4K gene and protein expression in ER+ human MCF7 breast cancer cells. Estrogen acts through ESR1 to regulate PRP4K expression, as over-expression of ESR1 in the ER-negative MDA-MB-231 breast cancer cell line increased the expression of this kinase, and knock-down of ESR1 in ER+ T47D breast cancer cells reduced PRP4K levels. Furthermore, treatment with 4-hydroxytamoxifen (4-OHT) resulted in a dose-dependent decrease in PRP4K protein expression in MCF7 cells. Consistent with our previous studies identifying PRP4K as a taxane-response biomarker, reduced PRP4K expression in 4-OHT-treated cells correlated with reduced sensitivity to paclitaxel. Thus, PRP4K is novel estrogen regulated kinase, and its levels can be reduced by 4-OHT in ER+ breast cancer cells altering their response to taxanes.

Loss of PRP4K drives anoikis resistance in part by dysregulation of epidermal growth factor receptor endosomal trafficking

Anoikis acts as a critical barrier to metastasis by inducing cell death upon cancer cell detachment from the extracellular matrix (ECM), thereby preventing tumor cell dissemination to secondary sites. The induction of anoikis requires the lysosomal-mediated downregulation of epidermal growth factor receptors (EGFRs) leading to termination of pro-survival signaling. In this study, we demonstrate that depletion of pre-mRNA splicing factor 4 kinase (PRP4K; also known as PRPF4B) causes dysregulation of EGFR trafficking and anoikis resistance. We also report a novel cytoplasmic localization of PRP4K at the late endosome, and demonstrate both nuclear and cytoplasmic localization in breast, lung and ovarian cancer tissue. Mechanistically, depletion of PRP4K leads to reduced EGFR degradation following cell detachment from the ECM and correlates with increased TrkB, vimentin and Zeb1 expression. As a result, PRP4K loss promotes sustained growth factor signaling and increased cellular resistance to anoikis in vitro and in a novel zebrafish xenotransplantation model of anoikis sensitivity, as well as increased metastasis in a mouse model of ovarian cancer. Thus, PRP4K may serve as a potential biomarker of anoikis sensitivity in ovarian and other epithelial cancers.