BackgroundNerves are key factors in prostate cancer (PCa), but the functional role of innervation in prostate cancer is poorly understood. PCa induced neurogenesis and perineural invasion (PNI), are associated with aggressive disease.MethodWe denervated rodent prostates chemically and physically, before orthotopically implanting cancer cells. We also performed a human neoadjuvant clinical trial using botulinum toxin type A (Botox) and saline in the same patient, before prostatectomy.ResultBilateral denervation resulted in reduced tumor incidence and size in mice. Botox treatment in humans resulted in increased apoptosis of cancer cells in the Botox treated side. A similar denervation gene array profile was identified in tumors arising in denervated rodent prostates, in spinal cord injury patients and in the Botox treated side of patients. Denervation induced exhibited a signature gene profile, indicating translation and bioenergetic shutdown. Nerves also regulate basic cellular functions of non‐neoplastic epithelial cells.ConclusionNerves play a role in the homeostasis of normal epithelial tissues and are involved in prostate cancer tumor survival. This study confirms that interactions between human cancer and nerves are essential to disease progression. This work may make a major impact in general cancer treatment strategies, as nerve/cancer interactions are likely important in other cancers as well. Targeting the neural microenvironment may represent a therapeutic approach for the treatment of human prostate cancer.

BACKGROUND Semaphorin 4F (S4F) has roles in embryological axon guidance and is expressed in adults. S4F is involved in cancer-induced neurogenesis. METHODS Prostate cells were transfected with S4F retrovirus. Cells and controls were used for a BrdU incorporation assay (proliferation) and in vitro scratch and matrigel transwell chamber invasion assay (migration). Monoclonal antibodies were developed using baculovirus expressed recombinant GST-S4F and used to immunostain tissue microarrays. Slides were imaged using deconvolution and analyzed using tissue segmentation. Data was correlated with clinico-pathological parameters, other biomarkers and survival analysis performed. Heterogeneity of S4F expression was analyzed with unsupervised clustering algorithms. RESULTS Proliferation rates measured by BrdU incorporation were higher in all S4F transfected cells. S4F over-expression was associated with increased motility of the cancer cells. S4F expression was over expressed in HGPIN/PCa than normal epithelium. S4F expression correlated with seminal vesicle invasion. Patients with high values of S4F in PCa cytoplasm are at significantly higher risk of biochemical recurrence, by univariate and multivariate analysis. S4F cytoplasmic expression in PCa cells also correlates with nerve density in PCa and perineural invasion diameter. Correlations were identified with NFkB and inversely with apoptosis in PNI. CONCLUSION This data demonstrates that S4F is significantly involved in human PCa progression. S4F is a key regulator of the interactions between nerves in the tumor microenvironment and cancer cells. Because of the importance of cancer nerve interaction in the biology of cancer and its clinical implication, S4F can be considered a major therapeutic target.

Cancer related axonogenesis and neurogenesis are recently described biologic phenomena. Our previously published data showed that nerve density and the number of neurons in the parasympathetic ganglia are increased in prostate cancer and associated with aggressive disease. Tissue microarrays were constructed from 640 radical prostatectomy specimens with prostate cancer. Anti-PGP 9.5 antibodies were used to identify and quantify nerve density. Protein expression was objectively analyzed using deconvolution imaging, image segmentation and image analysis. Data was correlated with clinico-pathological variables and tissue biomarkers available in our database. Nerve density, as measured by PGP 9.5 expression, had a weak but significant positive correlation with the lymph node status (rho=0.106; p=0.0275). By Cox univariate analysis, PGP 9.5 was a predictor of time to biochemical recurrence, but not on multivariate analysis. Increased nerve density correlated with increased proliferation of prostate cancer cells. It also correlated with expression of proteins involved in survival pathways (p-Akt, NFKB, GSK-2, PIM-2, CMYC, SKP-2, SRF, P27n, PTEN), with increased levels of hormonal regulation elements (AR, ER Alpha), and co-regulators and repressors (SRC-1, SRC-2, AIB-1, DAX). Axonogenesis is a recently described phenomenon of paramount importance in the biology of prostate cancer. While the degree of axonogenesis is predictive of aggressive behavior in prostate cancer, it does not add to the information present in current models on multivariate analysis. We present data that corroborates that axonogenesis is involved in biological processes such as proliferation of prostate cancer, through activation of survival pathways and interaction with hormonal regulation.

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Perineural invasion is a symbiotic relationship between cancer cells and nerves and is most frequently seen in “neurotropic” cancers, such as prostate cancer. It results in increased perineural space cancer cell growth and decreased apoptosis and induces nerve growth. Tissue microarrays (TMA) were constructed from 640 radical prostatectomy specimens with prostate cancer. The perineural diameter was measured as previously described. Multiple biomarkers have been previously performed on this TMA cohort and all data was kept in the same database. The biomarker results database was queried for correlations between perineural invasion diameter and tissue biomarkers. Increased perineural invasion diameter correlated with increased proliferation of prostate cancer cells, and with apoptosis. It also correlated with proteins involved in survival pathways such as NFkB, C-MYC, phosphorylated AKT and its downstream effector FHKR, but not with GSK. Unlike nerve density it did not correlate with decreased PTEN expression. Increased perineural invasion diameter was associated with higher levels of hormonal receptors such as androgen receptor, but not estrogen receptor. Also associated with perineural invasion diameter were co-regulators and co-repressors including SRC1 and TIF2. Perineural invasion diameter had the strongest correlation with tumor volume (rho=0.579; p=0.000), not identified with nerve density. These data demonstrate that perineural invasion has similar biologic correlations as neural density. However, we found a distinct and very strong correlation with increased tumor volume. This data confirms that perineural invasion is the ultimate and most successful interaction between cancer cells and nerve fibers, resulting in increased tumor growth.

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