Elucidation of the functions of Neuropilin 2 in osteoclasts in promoting prostate cancer bone metastasis.

Bone metastasis is one of the major clinical concerns that causes skeletal related malignancies and increased mortality. Bone is one of the preferred sites for metastatic prostate cancer. The metastatic prostate cancer cells interact with bone cells (osteoblasts and osteoclasts) resulting in an imbalance in the bone homeostasis leading to increased activation of osteoblasts over osteoclasts. Our preliminary data indicated a non-tyrosine kinase receptor Neuropilin 2 (NRP2) is expressed in osteoclasts induced by metastatic prostate cancer cells and acts as a negative regulator of osteoclast differentiation and function. We hypothesize that prostate cancer -induced NRP2 expression in osteoclasts is necessary for low osteolytic activity and thus favors an osteoblastic lesion in prostate cancer bone metastasis. Early experimentation discussed in my first section of my thesis demonstrated an increase in NRP2 expression in osteoclasts induced by RANKL and M-CSF and in PC3 and LNCaP C4-2B conditioned media (CM). TRAP staining and activity confirmed the differentiation of osteoclasts under these conditions. Interestingly, depletion of NRP2 and treatment with either in RANKL and M-CSF or LNCaP C4-2B CM exhibited a drastic increase in osteoclast differentiation and function. An increase in expression of osteoclastic genes following NRP2 depletion in RANKL and M-CSF and LNCaP C4-2B CM was also detected. However, NRP2-depleted osteoclast precursors when treated with PC3 CM showed no change in osteoclastogenesis. It is important to note that LNCaP C4-2B promotes mixed bone lesions, which inclines more toward osteoblastic lesion, while PC3 promotes predominantly osteoclastic bone lesions. These findings therefore advocate a role of NRP2 in inhibiting osteoclastic activity in PCa bone metastasis with mixed lesions and that osteolytic PCa evades NRP2 inhibition. In the second section of my dissertation, we elucidated the molecular mechanisms through which NRP2 regulates osteoclast differentiation and function in normal bone and in LNCaP C4-2B CM. Our studies suggest that NRP2 regulates the expression and translocation of NFATC1 which is a crucial osteoclastic transcription factor. Additionally, NRP2 controls NF-κB in the normal bone. These data imply that NRP2 restricts the translocation of critical transcription factors to regulate osteoclasts in prostate cancer bone metastasis. The last part of my dissertation addressed how PC3 CM-induced OCs escapes the inhibition of NRP2. Presence of GM-CSF resulted in a delay in the differentiation and fusion of osteoclasts in RANKL and M-CSF and LNCaP C4-2B CM. It can be deduced that secretion of GM-CSF by PC3 CM may regulate the differentiation and fusion of osteoclasts and thereby escapes the regulation of NRP2. Altogether, we report that NRP2 functions as a negative regulator of osteoclasts in prostate cancer bone metastasis but is rendered ineffective in osteolytic lesions. Hence, an insight into the regulation of NRP2 in osteoclasts can aid in the development of new and effective therapeutic strategies for the treatment of prostate cancer bone metastasis

NRP2 transcriptionally regulates its downstream effector WDFY1.

Neuropilins (NRPs) are cell surface glycoproteins that often act as co-receptors for plexins and VEGF family receptors. Neuropilin-2 (NRP2), a family member of NRPs, was shown to regulate autophagy and endocytic trafficking in cancer cells, a function distinctly different from its role as a co-receptor. WD Repeat and FYVE domain containing 1 (WDFY1)-protein acts downstream of NRP2 for this function. Our results indicated that NRP2 maintains an optimum concentration of WDFY1 by negatively regulating its expression. Since increased expression of WDFY1 reduces the endocytic activity, maintenance of WDFY1 level is crucial in metastatic cancer cells to sustain high endocytic activity, essential for promotion of oncogenic activation and cancer cell survival. Here, we have delineated the underlying molecular mechanism of WDFY1 synthesis by NRP2. Our results indicated that NRP2 inhibits WDFY1 transcription by preventing the nuclear localization of a transcription factor, Fetal ALZ50-reactive clone 1 (FAC1). Our finding is novel as transcriptional regulation of a gene by NRP2 axis has not been reported previously. Regulation of WDFY1 transcription by NRP2 axis is a critical event in maintaining metastatic phenotype in cancer cells. Thus, inhibiting NRP2 or hyper-activating WDFY1 can be an effective strategy to induce cell death in metastatic cancer

Tumor- and Osteoclast-Derived NRP2 in Prostate Cancer Bone Metastases

Understanding the role of neuropilin 2 (NRP2) in prostate cancer cells as well as in the bone microenvironment is pivotal in the development of an effective targeted therapy for the treatment of prostate cancer bone metastasis. We observed a significant upregulation of NRP2 in prostate cancer cells metastasized to bone. Here, we report that targeting NRP2 in cancer cells can enhance taxane-based chemotherapy with a better therapeutic outcome in bone metastasis, implicating NRP2 as a promising therapeutic target. Since, osteoclasts present in the tumor microenvironment express NRP2, we have investigated the potential effect of targeting NRP2 in osteoclasts. Our results revealed NRP2 negatively regulates osteoclast differentiation and function in the presence of prostate cancer cells that promotes mixed bone lesions. Our study further delineated the molecular mechanisms by which NRP2 regulates osteoclast function. Interestingly, depletion of NRP2 in osteoclasts in vivo showed a decrease in the overall prostate tumor burden in the bone. These results therefore indicate that targeting NRP2 in prostate cancer cells as well as in the osteoclastic compartment can be beneficial in the treatment of prostate cancer bone metastasis

Association of HIF-1α with p300 in the presence of FIH-1, PTEN in U87 cells.

<p>A: Western blot of HIF-1α from protein lysates of U87 cells incubated overnight either in hypoxic (3% oxygen) or normoxic (21% oxygen) conditions (top panel). No change of p300 protein levels was observed in the same samples (middle panel). β-actin was used as a loading control (lower panel). B: Association of HIF-1α with p300 in U87 cells: cells were transiently transfected with either FIH-1 or a PTEN expression plasmid, or both of them together for 24 hrs, and then incubated overnight under both normoxic and hypoxic conditions. Nuclear extracts were collected and subjected to immunoprecipitation with anti-p300 antibody followed by Western blot analysis with anti-HIF-1α (top panel) and IgG (middle panel). Nuclear extracts were also subjected to western blot analysis with anti- HIF-1α antibody (bottom panel).</p

The Role of Factor Inhibiting HIF (FIH-1) in Inhibiting HIF-1 Transcriptional Activity in Glioblastoma Multiforme

<div><p>Glioblastoma multiforme (GBM) accounts for about 38% of primary brain tumors in the United States. GBM is characterized by extensive angiogenesis induced by vascular growth factors and cytokines. The transcription of these growth factors and cytokines is regulated by the <u>H</u>ypoxia-<u>I</u>nducible-<u>F</u>actor-1(HIF-1), which is a key regulator mediating the cellular response to hypoxia. It is known that <u>F</u>actor <u>I</u>nhibiting <u>H</u>IF-1, or FIH-1, is also involved in the cellular response to hypoxia and has the capability to physically interact with HIF-1 and block its transcriptional activity under normoxic conditions. Delineation of the regulatory role of FIH-1 will help us to better understand the molecular mechanism responsible for tumor growth and progression and may lead to the design of new therapies targeting cellular pathways in response to hypoxia. Previous studies have shown that the chromosomal region of 10q24 containing the FIH-1 gene is often deleted in GBM, suggesting a role for the FIH-1 in GBM tumorigenesis and progression. In the current study, we found that FIH-1 is able to inhibit HIF-mediated transcription of GLUT1 and VEGF-A, even under hypoxic conditions in human glioblastoma cells. FIH-1 has been found to be more potent in inhibiting HIF function than PTEN. This observation points to the possibility that deletion of 10q23-24 and loss or decreased expression of FIH-1 gene may lead to a constitutive activation of HIF-1 activity, an alteration of HIF-1 targets such as GLUT-1 and VEGF-A, and may contribute to the survival of cancer cells in hypoxia and the development of hypervascularization observed in GBM. Therefore FIH-1 can be potential therapeutic target for the treatment of GBM patients with poor prognosis.</p></div

The mRNA levels of GLUT-1 were reduced with FIH-1 overexpression.

<p>1A and 1B: U87 cells were transfected with FIH-1-expression plasmid and cultured in both hypoxic (1B) and normoxic (1A) conditions. Total RNA was harvested and RQ-PCR was performed using primers specific for GLUT-1 and 36B4 (internal control). A significant decrease of GLUT-1 mRNA levels was observed in cells transfected with FIH-1 expression plasmid in both normoxia and hypoxia. The data here is the mean from three independent results. * Represents p value less than 0.05. 1C and 1D: The expression levels of GLUT-1 were decreased by overexpression of FIH-1 in U87 cells. U87 cells were transfected with constructs either expressing FIH-1 or with vector alone and incubated for 48 hrs under normoxic condition. Cells were collected and subjected to flow cytometry analysis. A reduction in GLUT-1 levels was detected in cells transfected with FIH-1 plasmid (1D) compared with cells transfected with control vector (1C), as shown in the P3 population.</p

Inhibition of VEGF-A expression by FIH-1.

<p>VEGF-A expression was controlled by FIH-1 in both normoxia and hypoxia. U87 cells were transfected with the FIH-1 expression plasmid at different doses (e.g. 0.5 µg/ml, 1.5 µg/ml) and cultured under both hypoxic and normoxic conditions. Total RNA was obtained and then RQ-PCR was performed using primers specific for VEGF-A and 36B4 (internal control). VEGF-A mRNA levels were decreased with FIH-1 overexpresssion under both normoxic and hypoxic conditions (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086102#pone-0086102-g004" target="_blank">Figure 4A</a>). FIH-1 mRNA levels were also measured as a control experiment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086102#pone-0086102-g004" target="_blank">Figure 4B</a>). The data here represent the mean of three independent experiments, and * represents p<0.05.</p

Inhibit GLUT-1 transcription by FIH-I in U87 under hypoxia GLUT-1 expression was inhibited by PTEN under normoxic condition.

<p>U87 cells were transfected with FIH-1 and PTEN expression plasmids either alone or in combination and cultured in both hypoxic and normoxic conditions. 2A: Total RNA was obtained and followed with RQ-PCR using primers specific for GLUT-1 and 36B4 (internal control). A significant reduction of GLUT-1 mRNA levels was observed in cells expressing PTEN, FIH-1, or both under normoxic conditions, but interestingly, GLUT-1 mRNA levels were not changed in cells expressing PTEN under hypoxic conditions. Average and standard deviation of three independent experiments were calculated. * Represents p value less than 0.05. 2B: Whole cell lysates from each experimental condition were collected and protein levels of FIH-1 (top panel) and PTEN (middle panel) were examined by Western blot. β-actin was used as a loading control (lower panel).</p