Anna M. Brown

Increasing Melanoma Cell Death Using Inhibitors of Protein Disulfide Isomerases to Abrogate Survival Responses to Endoplasmic Reticulum Stress

Exploiting vulnerabilities in the intracellular signaling pathways of tumor cells is a key strategy for the development of new drugs. The activation of cellular stress responses mediated by the endoplasmic reticulum (ER) allows cancer cells to survive outside their normal environment. Many proteins that protect cells against ER stress are active as protein disulfide isomerases (PDI) and the aim of this study was to test the hypothesis that apoptosis in response to ER stress can be increased by inhibiting PDI activity. We show that the novel chemotherapeutic drugs fenretinide and velcade induce ER stress-mediated apoptosis in melanoma cells. Both stress response and apoptosis were enhanced by the PDI inhibitor bacitracin. Overexpression of the main cellular PDI, procollagen-proline, 2-oxoglutarate-4-dioxygenase beta subunit (P4HB), resulted in increased PDI activity and abrogated the apoptosis-enhancing effect of bacitracin. In contrast, overexpression of a mutant P4HB lacking PDI activity did not increase cellular PDI activity or block the effects of bacitracin. These results show that inhibition of PDI activity increases apoptosis in response to agents which induce ER stress and suggest that the development of potent, small-molecule PDI inhibitors has significant potential as a powerful tool for enhancing the efficacy of chemotherapy in melanoma.

c-abl is involved in the association of p53 and trk A.

The p53 tumour suppressor phosphoprotein associates with proteins involved in DNA replication, transcription, cell cycle machinery and regulation of its own expression. Recently it has been shown that p53 can also bind to trk A tyrosine kinase which is the receptor for nerve growth factor (NGF). This study demonstrates that p53 appears to associate with trk A via c-abl. Endogenous c-abl was detected when the trk A and p53 complex was immunoprecipitated from lysates of NGF stimulated NIH3T3 cells expressing trk A or NIH3T3 cells expressing trk A and a temperature sensitive p53 (val 135). Endogenous c-abl and trk A association was observed in NGF stimulated p53 negative fibroblasts transfected with trk A alone; suggesting that c-abl can independently bind to trk A in the absence of p53. Interestingly, association between endogenous p53 and trk A was not detected in NGF stimulated abl negative fibroblasts transfected with trk A or when these cells were exposed to gamma radiation. This result suggests that p53 preferentially binds to trk A in the presence of c-abl and that p53 and trk A do not appear to associate directly even if p53 is activated and its levels increased by gamma radiation. Overall, these data suggest that c-abl is possibly acting as an adaptor or bridge between p53 and trk A.

Lysophosphatidic Acid Promotes Cell Migration through STIM1- and Orai1-Mediated Ca2+i Mobilization and NFAT2 Activation

Lysophosphatidic acid (LPA) enhances cell migration and promotes wound healing in vivo, but the intracellular signaling pathways regulating these processes remain incompletely understood. Here we investigated the involvement of agonist-induced Ca2+ entry and STIM1 and Orai1 proteins in regulating nuclear factor of activated T cell (NFAT) signaling and LPA-induced keratinocyte cell motility. As monitored by Fluo-4 imaging, stimulation with 10 μℳ LPA in 60 μℳ Ca2+o evoked Ca2+i transients owing to store release, whereas addition of LPA in physiological 1.2 mℳ Ca2+o triggered store release coupled to extracellular Ca2+ entry. Store-operated Ca2+ entry (SOCE) was blocked by the SOCE inhibitor diethylstilbestrol (DES), STIM1 silencing using RNA interference (RNAi), and expression of dominant/negative Orai1R91W. LPA induced significant NFAT activation as monitored by nuclear translocation of green fluorescent protein-tagged NFAT2 and a luciferase reporter assay, which was impaired by DES, expression of Orai1R91W, and inhibition of calcineurin using cyclosporin A (CsA). By using chemotactic migration assays, LPA-induced cell motility was significantly impaired by STIM1, CsA, and NFAT2 knockdown using RNAi. These data indicate that in conditions relevant to epidermal wound healing, LPA induces SOCE and NFAT activation through Orai1 channels and promotes cell migration through a calcineurin/NFAT2-dependent pathway.

Mechanistic and functional changes in Ca2+ entry after retinoic acid-induced differentiation of neuroblastoma cells

We have investigated effects of neuronal differentiation on hormone-induced Ca2+ entry. Fura-2 fluorescence measurements of undifferentiated SH-SY5Y neuroblastoma cells, stimulated with methacholine, revealed the presence of voltage-operated Ca2+-permeable, Mn2+-impermeable entry pathways, and at least two voltage-independent Ca2+- and Mn2+-permeable entry pathways, all of which apparently contribute to both peak and plateau phases of the Ca2+ signal. Similar experiments using 9-cis retinoic acid-differentiated cells, however, revealed voltage-operated Ca2+-permeable, Mn2+-impermeable channels, and, more significantly, the absence or down-regulation of the most predominant of the voltage-independent entry pathways. This down-regulated pathway is probably due to CCE (capacitative Ca2+ entry), since thapsigargin also stimulated Ca2+ and Mn2+ entry in undifferentiated but not differentiated cells. The Ca2+ entry components remaining in methacholine-stimulated differentiated cells contributed to only the plateau phase of the Ca2+ signal. We conclude that differentiation of SH-SY5Y cells results in a mechanistic and functional change in hormone-stimulated Ca2+ entry. In undifferentiated cells, voltage-operated Ca2+ channels, CCE and NCCE (non-CCE) pathways are present. Of the voltage-independent pathways, the predominant one appears to be CCE. These pathways contribute to both peak and plateau phases of the Ca2+ signal. In differentiated cells, CCE is either absent or down-regulated, whereas voltage-operated entry and NCCE remain active and contribute to only the plateau phase of the Ca2+ signal.

Analysis of trk A and p53 association.

trk A tyrosine kinase (the high affinity receptor for nerve growth factor) binds to the p53 tumour suppressor protein in vitro and in vivo. Our aim was to determine which regions of p53 are involved in trk A association. In vitro binding experiments using baculovirus expressed trk A and in vitro transcribed and translated C‐terminus p53 deletion mutants show amino acids 327–338 critical for association. Also, analysis with mutants at the N‐terminus, conserved regions II, III, IV and V or amino acid positions 173, 175, 181, 248 and 249 (which are amino acids frequently mutated in a variety of neoplasms and transformed cell lines), show that these sites are not involved in trk A binding. Importantly, similar results are obtained after immunoprecipitation of lysates from p53 negative fibroblasts expressing trk A and the above p53 mutant proteins. These data suggest that the amino‐terminus of the oligomerisation domain of p53 is involved in p53/trk A association.

Abstract CT222: Ferumoxytol enhanced MRI for lymph node staging in genitourinary cancers

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: Conventional imaging has limited accuracy in genitourinary (GU) cancer staging. This study examines the utility of ferumoxytol enhanced MRI in lymph node (LN) staging of GU cancers. Methods: This ongoing IRB-approved phase II clinical trial enrolls patients with prostate cancer, renal cell carcinoma, or bladder cancer at high risk for LN metastases. Patients undergo baseline T2 and T2* weighted MRI scans followed by injection of 7.5mg/Kg ferumoxytol. Repeat scans are acquired at 24hr and 48hr post-injection. The criterion for positive LNs was preservation of hyper-intense signal indicating failure to take up ferumoxytol. Validation was by histopathology when available or on clinical grounds, for which LNs that changed size on routine imaging were considered true positives. Results: To date, 13 patients have completed the study. Of 11 prostate cancer patients, one was studied pre-operatively while 10 had suspected therapy failure. Median age and PSA were 65yrs (36-75) and 5.6ng/mL (0.3-201). The other 2 patients had renal cell carcinoma and bladder cancer. Overall, 20 LNs were identified with mean size 1.9cm (0.7-3.8) long axis by 1.3cm (0.6-2.6) short axis. There were 14 true positive LNs, 1 false positive, 1 false negative, and 4 nodes pending validation. Validation was by histopathology for 7 LNs, with 2 nodes pending biopsy, and clinical grounds for 13 LNs, with 2 inconclusive nodes awaiting further validation. Ferumoxytol correctly identified LN status in 9 of 10 patients with validated nodes (Table 1). Conclusions: Ferumoxytol enhanced MRI shows promise in detecting malignant LNs >6mm in GU cancer patients. Since the method involves a conventional MRI unit with off-label use of an FDA-approved agent, it could be widely available. However, further validation is necessary before routine use. Table 1: Preliminary results for LN staging in GU cancer patients using ferumoxytol enhanced MRI | Subject | Study Arm | Gender | Age (yr) | PSA at study initiation (ng/mL) | LN number | LN location | size/long axis (cm) | size/short axis (cm) | Ferumoxytol positive? 1 = yes, 0 = no | Result | |:------- | -------------------- | ------ | -------- | ------------------------------- | --------- | -------------- | ------------------- | -------------------- | ------------------------------------- | ------------ | | 1 | prostate cancer | M | 63 | 25.06 | 1 | R ext iliac | 3.0 | 2.6 | 1 | TP | | | | | | | 2 | L ext iliac | 1.3 | 0.8 | 1 | TP | | 2 | prostate cancer | M | 65 | 73.96 | 1 | L RP | 1.6 | 1.4 | 1 | TP | | | | | | | 2 | L int iliac | 3.8 | 1.9 | 1 | TP | | 3 | prostate cancer | M | 64 | 10.49 | 1 | R ext iliac | 3.0 | 0.9 | 1 | pending | | | | | | | 2 | L ext iliac | 1.9 | 1.1 | | pending | | 4 | prostate cancer | M | 74 | 2.06 | 1 | L RP | 1.7 | 1.5 | 1 | TP | | 5 | prostate cancer | M | 64 | 2.89 | 1 | R ext iliac | 1.6 | 1.1 | 1 | TP | | 6 | prostate cancer | M | 65 | 0.28 | 1 | L int iliac | 1.5 | 0.8 | 1 | TP | | 7 | prostate cancer | M | 75 | 2.87 | 1 | R int iliac | 0.7 | 0.7 | 1 | TP | | 8 | prostate cancer | M | 64 | 27.91 | 1 | R common iliac | 1.7 | 1.7 | 1 | inconclusive | | 9 | prostate cancer | M | 72 | 201.2 | 1 | L RP | 2.3 | 1.7 | | FN | | 10 | prostate cancer | M | 73 | 6.77 | 1 | L int iliac | 1.5 | 1.0 | 1 | TP | | 11 | prostate cancer | M | 36 | 1.35 | 1 | R femoral | 0.8 | 0.8 | 1 | FP | | | | | | | 2 | L ext iliac | 1.5 | 1.0 | 1 | inconclusive | | | | | | | 3 | R perirectal | 0.8 | 0.6 | 1 | TP | | 12 | renal cell carcinoma | F | 41 | N/A | 1 | aortocaval | 2.8 | 2.2 | 1 | TP | | | | | | | 2 | R RP | 3.6 | 2.3 | 1 | TP | | | | | | | 3 | R int iliac | 2.0 | 1.5 | 1 | TP | | 13 | bladder cancer | M | 59 | N/A | 1 | aortocaval | 1.5 | 1.0 | 1 | TP | RP = retroperitoneal, TP = true positive, FN = false negative, FP = false positive, ext = external, int = internal Citation Format: Anna M. Brown, Sandeep Sankineni, Marcelino Bernardo, Dagane Daar, Juanita Weaver, Yolanda McKinney, Anna Couvillon, James L. Gulley, Bradford J. Wood, Peter A. Pinto, William L. Dahut, Ravi Amrit Madan, Peter L. Choyke, Baris Turkbey. Ferumoxytol enhanced MRI for lymph node staging in genitourinary cancers. [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 CT222. doi:10.1158/1538-7445.AM2015-CT222

Lysophosphatidic acid promotes keratinocyte migration through STIM1-regulated Orai1-Ca2+ channels and NFAT2 activation

S | Growth Factors and Signal Transduction