Daniel Djakiew

Distribution of Nerve Growth Factor-Like Protein and Nerve Growth Factor Receptor in Human Benign Prostatic Hyperplasia and Prostatic Adenocarcinoma

Recent observations from our laboratory have identified a nerve growth factor (NGF)-like protein in conditioned media of stromal cells and neoplastic epithelial cells of the human prostate which mediates paracrine interactive growth of both cell types in vitro. In order to investigate the location of this NGF-like protein in the human prostate in vivo, and whether a nerve growth factor receptor (NGF-R) could be identified, we have carried out immunocytochemical studies on frozen tissue sections of human benign prostatic hyperplasia (BPH), prostatic adenocarcinoma and normal prostatic tissue. The NGF-like protein localized predominantly to the stromal component of BPH, adenocarcinoma and normal (non-cancerous) prostatic tissue. Conversely, the NGF-R localized predominantly to the epithelial cells of these tissues. Renal tissue provided negative controls for both the NGF-like protein and the NGF-R. The testis provided positive controls for both the NGF-like protein and the NGF-R. These results provide corroborative evidence for a NGF-like protein produced by stromal cells which interacts with a NGF-R on the adjacent epithelial cells thereby mediating paracrine interactive growth regulation of the human prostate.

Molecular characterization of neurotrophin expression and the corresponding tropomyosin receptor kinases (trks) in epithelial and stromal cells of the human prostate.

The prostate is one of the most abundant sources of nerve growth factor (NGF) outside of the nervous system. NGF is a member of the neurotrophin family of growth factors which in mammals also includes brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5). These neurotrophins can bind with high affinity to a family of tropomyosin receptor kinases (trks). These receptors are trkA, which binds NGF; trkB, which binds both BDNF and NT-4/5; and trkC, which binds NT-3. In order to characterize the molecular expression of the neurotrophins and their corresponding trk receptors in the prostate we performed Northern blot analysis for the neurotrophins and reverse transcription-polymerase chain reaction (RT-PCR) coupled with Southern blot analysis for the trk family of receptors on smooth muscle stromal cells from the prostate, the androgen responsive LNCaP prostate tumor cell line and the androgen refractory TSU-pr1 prostate tumor cell line. The results show that smooth muscle stromal cells expressed NGF, BDNF and trkC, whereas both epithelial cell lines expressed trkA, trkB and trkC to various degrees. NT-3 was not detected in either the smooth muscle stromal cells or in both epithelial cell lines. This suggests that the stromal cell derived NGF and BDNF may interact via paracrine mechanisms with trkA and trkB receptors, respectively, on the adjacent epithelial cells. Interestingly, the androgen responsive LNCaP cell line did not express any of the neurotrophins, whereas the androgen refractory TSU-pr1 cell line expressed NGF, BDNF and NT-4/5. This suggests that the autocrine expression of NGF, BDNF and NT-4/5 is up-regulated in prostate epithelial cells following their transformation to an androgen refractory pathology. Hence, the malignant transformation of prostate epithelial tumor cells may facilitate their escape from a paracrine dependence on stromal cell derived neurotrophins by the acquisition of the autocrine expression of neurotrophins. Since the pathology of malignant cell migration within the prostate is predominantly by direct extension around prostatic nerves the upregulation of autocrine neurotrophin expression within prostate epithelial tumor cells may be concomitant with transformation to a malignant phenotype capable of invasion along the perineural space and extracapsular metastasis to distant sites of tumor formation.

Epidermal growth factor (EGF) promotes chemomigration of a human prostate tumor cell line, and EGF immunoreactive proteins are present at sites of metastasis in the stroma of lymph nodes and medullary bone.

Prostate tumor cells preferentially metastasize to bony sites and lymph nodes at a frequency in excess of that which would be predicted by random tumor cell dissemination. In order to determine whether chemoattractants in these organs promote organ‐specific metastasis, we utilized human cell lines derived from and/or related to these organs as sources of potential chemoattractants. Secretory proteins derived from the cell lines MG‐63 (osteosarcoma), SK‐ES‐1 (Ewings sarcoma), and KG‐1 (leukemia) stimulated chemomigration of the TSU‐pr1 prostate tumor cells in a dose‐dependent manner in Boyden chambers. In addition, secretory proteins from a human prostatic stromal cell line (hPS) and from the TSU‐Pr1 prostate tumor cell line were also able to stimulate chemomigration of the TSU‐pr1 cells through Boyden chambers. Since lymph nodes and bony sites represent organs of hematopoietic/lymphoid proliferation and activation, we undertook identification of specific cytokines present at these sites which may promote the chemomigration of prostate tumor cells. In this context, the cytokines interleukin‐1α, interleukin‐2, interleukin‐6, tumor necrosis factor‐β, transforming growth factor‐β, interferon α2‐a, and granulocyte‐macrophage colony‐stimulating factor did not stimulate chemomigration of the TSU‐pr1 prostate tumor cell line. In contrast, the cytokine epidermal growth factor (EGF) stimulated chemomigration of the TSU‐pr1 prostate tumor cells through the Boyden chambers in a dose‐dependent manner. Western blot analysis of secretory proteins from the cell lines KG‐1, SK‐ES‐1, MG‐63, hPS, and TSU‐pr1 identified EGF‐immunoreactive proteins in all cases. In addition, EGF immunoreactivity was localized to the stroma of the human prostate, the osteogenic stroma of pelvic medullary bone, and the stroma within the capsule and trabeculae of pelvic lymph nodes. Hence, these results demonstrate that the cytokine EGF promotes the chemomigration of the TSU‐pr1 prostate tumor cell line, and that EGF within the stroma of pelvic lymph nodes and medullary bone may act as a chemoattractant for prostate tumor cells, thereby facilitating the preferential formation of metastatic foci within these organs.

Loss of low-affinity nerve growth factor receptor during malignant transformation of the human prostate

The low‐affinity nerve growth factor receptor (LNGFR) exhibits an inverse association of epithelial expression with the degree of differentiation of prostate adenocarcinoma tissue. However, the stage at which loss of LNGFR expression is first manifested in the malignant prostate has not been determined.

The p75NTR tumor suppressor induces caspase-mediated apoptosis in bladder tumor cells

p75NTR was identified as a tumor and metastasis suppressor that functions in part via induction of apoptosis in tumor cells. To examine p75NTR‐dependent apoptosis in tumor cells, we demonstrated that a dose‐dependent increase in p75NTR expression was associated with a concomitant increase in the mitochondrial proapoptotic effector proteins Bad, Bax and Bik and a decrease in the mitochondrial prosurvival effector proteins phospho‐Bad, Bcl‐2 and Bcl‐xL. Significantly, p75NTR‐dependent induction of cytochrome c release from the mitochondria occurred during CHX potentiation of apoptosis. Furthermore, p75NTR expression largely suppressed expression of IAP‐1 and induced cleavage of procaspase‐9 and procaspase‐7 but not of procaspases 2, 3, 6, 8 and 10. A specific peptide inhibitor of procaspase‐9 cleavage also inhibited cleavage of procaspase‐7, indicating that caspase‐7 is downstream of caspase‐9. As end points of apoptosis, we observed p75NTR‐dependent annexin V binding to the plasma membrane, an indicator of early apoptotic events, and Hoechst staining of DNA nuclear fragmentation, an indicator of late apoptotic events, whereas control tumor cells that lack expression of the p75NTR protein did not exhibit either of these apoptotic markers. Together, these results delineate the mitochondria‐mediated apoptotic pathway of the p75NTR tumor‐suppressor gene product.

Inhibition of chemomigration of a human prostatic carcinoma cell (TSU-pr1) line by inhibition of epidermal growth factor receptor function.

Chemoattractants expressed at bony sites and pelvic lymph nodes are thought to promote the preferential metastasis of human prostate tumor cells to these organs. Epidermal growth factor (EGF) is a potent chemoattractant for several human metastatic prostate tumor cell lines, including the TSU‐pr1 cell line, and EGF has been localized to the stroma of both bony sites and pelvic lymph nodes in humans. Hence, we investigated whether the TSU‐pr1 cell line expresses a functional EGF receptor (EGFR), which when antagonized reduces EGF‐mediated chemomigration of this cell line. In this context, the EGFR immunoprecipitated from cell lysates of TSU‐pr1 cells comigrated with the EGFR from A431 cells at a molecular weight of 170 kD. Addition of human EGF (hEGF) to the TSU‐pr1 cells for 5 min stimulated the dose‐dependent biphasic phosphorylation of the EGFR, with maximal stimulation of EGFR phosphorylation occurring at 2 ng/ml hEGF. In addition, treatment of hEGF‐stimulated (2 ng/ml) TSU‐pr1 cells with 0.5 μg/ml anti‐hEGFR monoclonal antibody or 100 nM staurosporine inhibited EGFR phosphorylation. Conversely, as negative controls, treatment of hEGF‐stimulated (2 ng/ml) TSU‐pr1 cells with K252a or dimethyl sulfoxide (DMSO) vehicle did not inhibit EGFR phosphorylation. TSU‐pr1 cells were stimulated to migration in 4 hr across Boyden chambers in response to 10 ng/ml hEGF. Treatment of the TSU‐pr1 cells with anti‐hEGFR monoclonal antibody inhibited in a dose‐dependent manner the chemomigration of the TSU‐pr1 cells across Boyden chambers. Similarly, treatment of the TSU‐pr1 cells with staurosporine inhibited in a dose‐dependent manner the chemomigration of the TSU‐pr1 cells across Boyden chambers. These results demonstrate that antagonists of hEGF‐mediated hEGFR phosphorylation also antagonize chemomigration of the TSU‐pr1 cells across Boyden chambers, suggesting that antagonists of the EGFR in prostate cancer may be useful in the treatment of metastatic disease.

The p75NTR metastasis suppressor inhibits urokinase plasminogen activator, matrix metalloproteinase-2 and matrix metalloproteinase-9 in PC-3 prostate cancer cells

The p75 neurotrophin receptor (p75NTR) has been characterized as a metastasis and tumor suppressor in prostate cancer. In order to investigate the mechanism(s) by which the p75NTR functions as a metastasis suppressor in prostate cancer cells, we characterized the ectopic expression of p75NTR on the urokinase plasminogen activator (uPA) and the type IV collagen matrix metalloproteinases (MMP-2 and MMP-9) in PC-3 human prostate cancer cells. Rank-order expression of p75NTR greatly reduced protein levels and enzymatic activities of uPA, MMP-2, and MMP-9 as shown by immunoblot and zymography analyses. Conversely, expression of the MMP-9 antagonist, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) exhibited an increase in protein levels with an increase in p75NTR levels, whereas TIMP-2 was not detected. Transient transfection with an inducible dominant negative antagonist Δp75NTR rescued uPA, MMP-2, and MMP-9 protein levels and protease activities, and conversely suppressed TIMP-1 levels. Since p75NTR signal transduction occurs via the NFκB and JNK pathways, antagonism of signaling intermediates in these pathways, using dominant negative IKKβ or dominant negative MKK-4, respectively, was shown to further decrease expression of uPA, MMP-2, and MMP-9 protein and enzymatic activity levels, and conversely up-regulate levels of TIMP-1. These results indicate that expression of uPA, MMP-2, MMP-9, and TIMP-1 are directly regulated by expression of p75NTR and its downstream signal transduction cascade. These results suggest that the metastasis suppressor activity of p75NTR is mediated, in part, by down-regulation of specific proteases (uPA, type IV collagenases) implicated in cell migration and metastasis.

Biochanin A reduces drug-induced p75NTR expression and enhances cell survival: a new in vitro assay for screening inhibitors of p75NTR expression.

Following spinal cord injury (SCI) or peripheral neuropathy, increased levels of the p75(NTR) death receptor initiate the signal transduction cascade leading to cell death. Investigations of compounds that may ameliorate neuronal cell death have largely used rodent models, which are time consuming, expensive, and cumbersome to perform. Previous studies had demonstrated that steroids, particularly dexamethasone and its analog methylprednisolone sodium succinate, exhibit limited neuroprotective effects against neuronal injury. Significantly, many naturally occurring nonsteroidal plant compounds exhibit structural overlap with steroids. In this report, we present an in vitro cellular screen model to practically examine the efficacy of various phytoestrogens in modulating the ibuprofen-induced expression of p75(NTR) and reduced cell survival of CCFSTTG1 and U87MG cells in a rescue (postinjury) or prevention (preinjury) regimen. We show that the phytoestrogen, biochanin A, and, to a lesser extent, genistein are more effective than dexamethasone at reducing p75(NTR) expression and improving the viability of U87MG and CCFSTTG1 before and after p75(NTR) induction. Furthermore, these studies implicate biochanin As inactivation of p38-MAPK as a possible contributor to reducing p75(NTR) with associated increased cell survival. This new in vitro assay facilitates a more time-efficient screening of compounds to suppress p75(NTR) expression and increase neuronal cell viability prior to their evaluation in animal models of neurological diseases.

The p38 MAPK Pathway in Prostate Cancer

The conventional signal transduction pathway for p38 MAPK is complex and diverse. A plethora of signals such as growth factors interact with death receptors to initiate a biochemical cascade by recruitment of activator molecules that in combination activate MAP3Ks. Many drugs intercede at the level of signal, activator, or MAP3Ks to mimic initiation of the signal transduction cascade. In the prostate, these signaling moieties, which include NSAIDs, converge at the level of MAP2Ks, ostensibly MKK6, which phosphorylates up to 4 isoforms of p38 MAPK. Phosphatases such as MKP1 or compounds such as biochanin A are able to antagonize activation of p38 MAPK. Phosphorylation of p38 MAPK allows phosphorylation of MK2 and MK3 that in turn promote stability of the p75NTR transcript. Concurrently, translocation of HuR from the nucleus to the cytoplasm and increased levels of HuR and eIF4E also promote p75NTR mRNA stability and increased levels of the p75NTR protein. In the prostate, the p75NTR functions as both a tumor and metastasis suppressor. In this context, increased expression of p75NTR modulates cell cycle effectors producing cytostasis in G0/G1, as well as mitochondrial effectors that modulate a caspase cascade leading to apoptosis. In addition, increased expression of p75NTR modulates motility effectors, ostensibly NAG-1, that retards cell migration. Hence, activation of the p38 MAPK pathway through a plethora of signal initiating events, leads to tumor and metastasis suppressor activity in prostate cancer cells.

Abstract 5271: Ibuprofen, an inhibitor of angiogenesis, enhances radiosensitivity and suppresses glioma invasion: Role of p75NTR and VEGF

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnCurrent treatments of malignant glioma, including anti-VEGF therapy, are limited by the invasive phenotype. Ionizing radiation is known to stimulate invasion, and p75NTR is a key regulator of glioma invasion. Because angiogenic inhibitors have been proposed as radiosensitizers, we hypothesized that ibuprofen (previously shown to regulate p75NTR) would serve as a radiosensitizer of glioma cells, and we studied its effect on glioma migration, invasion, and the expression of p75NTR and VEGF. The purpose of this study was to investigate the effect of combining radiation with ibuprofen on expression of p75NTR in human (U87, U251) and murine (GL261) glioma cells, and link to radiosensitivity, migration and invasion, and angiogenesis.nnMethods: We determined radiosensitivity using a clonogenic assay of glioma cells, including clones that were transfected to stably overexpress p75NTR. Glioma cells transfected with empty vector (pcDNA) were used as a control. Glioma migration and invasion were determined, respectively, by a wound scratch assay and Matrigel-Boyden chamber assay. Expression levels of p75NTR and VEGF165 were determined by Western blot analysis.nnResults: Ibuprofen increased the radiosensitivity of glioma cells as evidenced by a decreased survival fraction in the clonogenic assay. U87+p75NTR demonstrated increased radiosensitivity compared to control (U87pcDNA). As expected, U87+p75NTR U251+p75NTR were highly invasive and migratory compared to U87pcDNA and U251pcDNA. At a dose of 10 Gy, migration was significantly inhibited (P=0.02) in pcDNA cells only. Induction of p75NTR with ibuprofen correlated to increased radiosensitivity of glioma cells. Paradoxically we found that increased expression of p75NTR by ibuprofen correlated to a reduction of invasion and migration. In addition, expression of VEGF was lower in U87+p75 compared to U87pcDNA. When U87+p75 was treated with ibuprofen alone, VEGF was undetectable, when treated with radiation alone VEGF levels were lower than control but detected, and no VEGF was detectable when the cells were treated with both ibuprofen and radiation. In contrast, expression of VEGF was increased when p75pcDNA glioma cells were treated with ibuprofen, and further increased when treated with radiation. However, adding ibuprofen and radiation resulted in undetectable VEGF.nnConclusion: Our results suggest that ibuprofen could be useful clinically as a radiosensitizer and/or an agent to inhibit glioma invasion, migration and angiogenesis. Furthermore, our preliminary data suggest that there may be a “VEGF-switch” modulated by p75NTR status. The VEGF-p75NTR link could be important to drug discovery and translation to clinical control of the glioma malignant phenotype. (Supported by Dept of Defense grant USAMRAA -RX4301803, The Greg Wohrle Fund of the FBTA, the Moffitt Cancer Center Foundation, The Ben and Catherine Ivy Foundation).nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5271.