PRONGF/NGF and nerve infiltration in prostate and breast cancer

Research Doctorate - Doctor of Philosophy (PhD)Nerve fibre infiltration has recently been demonstrated as paramount to the tumor microenvironment and essential to cancer progression. However, it has not been clearly elucidated what attracts neuronal fibres into tumors. A plausible explanation is the overexpression of neurotrophic factors, such as nerve growth factor (NGF) or its precursor proNGF. These proteins are distinctively involved in neuronal survival/apoptosis through interactions with specific membrane receptors such as TrkA, p75^NTR and sortilin. This thesis aims to show whether proNGF/NGF overexpression drives nerve infiltration in prostate and breast cancer. What has been showed herein is that the overexpression of proNGF in a cohort of 104 prostate cancer cases, observed using immunohistochemistry has a direct correlation with the aggressiveness of prostate cancer (t_B= 0.51). A 60-kilodalton proNGF was detected by western blotting of prostate cancer cells, whereas the proteolytically processed NGF was not detected. In addition, we performed an in vitro co-culture of prostate cancer cells with neuronal cells and demonstrated the neurotrophic effect of prostate cancer cells to stimulate axonogenesis via the secretion of proNGF. Furthermore, in breast cancer we have shown nerve fibre infiltration by immunohistochemistry using the neuronal marker protein gene product (PGP) 9.5. Nerve infiltration was found to be associated with NGF expression and lymph node invasion. Secreted NGF was detected by dot blot analysis of conditioned medium from breast cancer cells. Interestingly, in vitro co-culture assays demonstrated that NGF secreted from breast cancer cells stimulated neurite outgrowth of neuronal cells, and that this effect could be inhibited by using an anti-NGF blocking antibody, thus supporting the neurotrophic potential of these cells. In conclusion, we have shown that proNGF is a driver of nerve infiltration in prostate cancer and that a similar phenomenon occurs in breast cancer via NGF. This study suggests new avenues for inhibiting prostate and breast cancer growth and metastasis by inhibiting axonogenesis via the targeting of proNGF and NGF

NGF and ProNGF: Regulation of neuronal and neoplastic responses through receptor signaling.

Nerve growth factor (NGF) and its precursor (proNGF) are primarily considered as regulators of neuronal function that induce their responses via the tyrosine kinase receptor TrkA and the pan-neurotrophin receptor p75NTR. It has been generally held that NGF exerts its effects primarily through TrkA, inducing a cascade of tyrosine kinase-initiated responses, while proNGF binds more strongly to p75NTR. When this latter entity interacts with a third receptor, sortilin, apoptotic responses are induced in contrast to the survival/differentiation associated with the other two. Recent studies have outlined portions of the downstream phosphoproteome of TrkA in the neuronal PC12 cells and have clarified the contribution of individual docking sites in the TrkA endodomain. The patterns observed showed a similarity with the profile induced by the epidermal growth factor receptor, which is extensively associated with oncogenesis. Indeed, as with other neurotrophic factors, the distribution of TrkA and p75NTR is not limited to neuronal tissue, thus providing an array of targets outside the nervous systems. One such source is breast cancer cells, in which NGF and proNGF stimulate breast cancer cell survival/growth and enhance cell invasion, respectively. This latter activity is exerted via TrkA (as opposed to p75NTR) in conjunction with sortilin. Another tissue overexpressing proNGF is prostate cancer and here the ability of cancer cells to induce neuritogenesis has been implicated in cancer progression. These studies show that the non-neuronal functions of proNGF/NGF are likely integrated with their neuronal activities and point to the clinical utility of these growth factors and their receptors as biomarkers and therapeutic targets for metastasis and cancer pain

Cxcr3-expressing leukocytes are necessary for neurofibroma formation in mice.

Plexiform neurofibroma is a major contributor to morbidity in patients with neurofibromatosis type I (NF1). Macrophages and mast cells infiltrate neurofibroma, and data from mouse models implicate these leukocytes in neurofibroma development. Antiinflammatory therapy targeting these cell populations has been suggested as a means to prevent neurofibroma development. Here, we compare gene expression in Nf1-mutant nerves, which invariably form neurofibroma, and show disruption of neuron-glial cell interactions and immune cell infiltration to mouse models, which rarely progresses to neurofibroma with or without disruption of neuron-glial cell interactions. We find that the chemokine Cxcl10 is uniquely upregulated in NF1 mice that invariably develop neurofibroma. Global deletion of the CXCL10 receptor Cxcr3 prevented neurofibroma development in these neurofibroma-prone mice, and an anti-Cxcr3 antibody somewhat reduced tumor numbers. Cxcr3 expression localized to T cells and DCs in both inflamed nerves and neurofibromas, and Cxcr3 expression was necessary to sustain elevated macrophage numbers in Nf1-mutant nerves. To our knowledge, these data support a heretofore-unappreciated role for T cells and DCs in neurofibroma initiation

ProNGF correlates with Gleason score and is a potential driver of nerve infiltration in prostate cancer

Nerve infiltration is essential to prostate cancer progression, but the mechanism by which nerves are attracted to prostate tumors remains unknown. We report that the precursor of nerve growth factor (proNGF) is overexpressed in prostate cancer and involved in the ability of prostate cancer cells to induce axonogenesis. A series of 120 prostate cancer and benign prostate hyperplasia (BPH) samples were analyzed by IHC for proNGF. ProNGF was mainly localized in the cytoplasm of epithelial cells, with marked expression in cancer compared with BPH. Importantly, the proNGF level positively correlated with the Gleason score (n = 104, tB = 0.51). A higher level of proNGF was observed in tumors with a Gleason score of =8 compared with a Gleason score of 7 and 6 (P < 0.001). In vitro, proNGF was detected in LNCaP, DU145, and PC-3 prostate cancer cells and BPH-1 cells but not in RWPE-1 immortalized nontumorigenic prostate epithelial cells or primary normal prostate epithelial cells. Co-culture of PC12 neuronal-like cells or 50B11 neurons with PC-3 cells resulted in neurite outgrowth in neuronal cells that was inhibited by blocking antibodies against proNGF, indicating that prostate cancer cells can induce axonogenesis via secretion of proNGF. These data reveal that ProNGF is a biomarker associated with high-risk prostate cancers and a potential driver of infiltration by nerves

SOD1 protein aggregates stimulate macropinocytosis in neurons to facilitate their propagation

Background Amyotrophic Lateral Sclerosis is characterized by a focal onset of symptoms followed by a progressive spread of pathology that has been likened to transmission of infectious prions. Cell-to-cell transmission of SOD1 protein aggregates is dependent on fluid-phase endocytosis pathways, although the precise molecular mechanisms remain to be elucidated. Results We demonstrate in this paper that SOD1 aggregates interact with the cell surface triggering activation of Rac1 and subsequent membrane ruffling permitting aggregate uptake via stimulated macropinocytosis. In addition, other protein aggregates, including those associated with neurodegenerative diseases (TDP-43, Htt ex1 46Q, α-synuclein) also trigger membrane ruffling to gain entry into the cell. Aggregates are able to rupture unstructured macropinosomes to enter the cytosol allowing propagation of aggregation to proceed. Conclusion Thus, we conclude that in addition to basic proteostasis mechanisms, pathways involved in the activation of macropinocytosis are key determinants in the spread of pathology in these misfolding diseases

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Additional file 10: of SOD1 protein aggregates stimulate macropinocytosis in neurons to facilitate their propagation

Characterization of iPSC derived motor neurons. (PDF 1487 kb