Influence of the neural microenvironment on prostate cancer

Abstract

Background: Nerves 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.

Method: We 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.

Result: Bilateral 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.

Conclusion: Nerves 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.

Keywords: Botox; cancer; denervation; nerves; neurogenesis; prostate.

Figure 1

Effects of denervation on histopathology of normal rat prostate and human prostate tumors. (A‐D) Morphology of non‐neoplastic rat prostate epithelium following MPG excision and Botox treatment. Panels A and B (hematoxylin and eosin [H&E], 200× and 600×, respectively) show the epithelium of the non‐neoplastic, neurally intact rat prostate. Panels C and D (H&E, 200× and 600×, respectively) indicate the generalized atrophy resulting from physical denervation and Botox treatment. (E) Chemical and physical denervation result in similar gene expression profiles. Cluster expression heat map of the top differentially expressed genes (ANOVA P < 0.01, SD > 0.2) in response to Botox or MPG treatment for both epithelial and stromal rat tissues. Rows, genes; columns, profiled samples. Within epithelial and stromal groups, genes are centered on the corresponding control. (F) Targeted metabolomic analysis of Botox‐treated tissues show significantly (FDR‐adjusted P‐value <0.10) reduced levels of metabolites associated with the TCA cycle and elevated levels of glucose/fructose and ribose.

Figure 2

(A and B) Tumors evolving in prostates with bilateral denervation with botox and MPG excision are significantly lower than controls (C) Evolving expression profiles of human prostate carcinoma cells growing in denervated rat prostates and in human patients. In the left panel, expression heat maps for genes regulated by both major pelvic ganglion (MPG) dissection and Botox (twofold each) in rats inoculated with VCaP human PCa cells are shown. For these same genes, the corresponding differential patterns are shown for laser‐captured carcinoma cells from the prostate of a spinal cord injury patient and the prostate of a patient with intact innervation (middle panel). In the right panel, gene expression profiles in a human PCa patient treated with saline or Botox. MPG/Botox profiles are centered on the corresponding control, whereas denervated versus intact prostate profiles are centered on the normal intact group. Yellow, high expression relative to reference; Blue, low expression. Results of physical and chemical denervation in mouse experiments: (D) Mouse VCaP‐luc IVIS luciferase luminosity detection at 3 weeks in the bilateral Botox denervation versus saline and spinal cord injury versus VCaP‐only control groups. (E) Luminosity detection at 6 weeks in the bilateral Botox denervation versus saline groups. (F) Overall prostate weight in the bilateral Botox denervation versus saline and spinal cord injury versus VCaP‐only control groups. (G) Castration improves the reduction of tumor size over bilateral botox (bilatera), but not over spinal cord injury.

Figure 3

(A) Design of the neoadjuvant human proof of principle clinical trial. Botox injected on the right side of the prostate and saline in the contralateral side. (B) Increased apoptotic ratio (TUNEL) in non‐neoplastic epithelium treated with Botox versus saline. (C) Similar increased in apoptotic ratio in PCa tissues treated with Botox versus saline. (D‐E) Nerve density was decreased in non‐neoplastic epithelial prostate tissues and cancer. (F‐G) Microvessel density in non‐neoplastic tissues and cancer. (H‐L) Morphologic changes induced by Botox treatment in human prostate tumors. In the example shown, a patient with bilateral Gleason stage 3 PCa was injected with Botox in the right prostate lobe, and saline in the left prostate lobe as an internal control. Panels J (H&E, 200×) and K (H&E, 600×) show the effects of Botox injection on the PCa tissues, including carcinoma cell involution with reduced cytoplasm and pyknotic nuclei, as compared to intact histology for Gleason 3 PCa observed in the contralateral lobe injected with saline (Panels H [H&E, 200×] and I [H&E, 600×]). (K) Nerve (Yellow arrowhead) surrounded by dead cancer cells with nuclear pyknosis and cytoplasmic disruption (H&E, 600×). (L) PCa cells treated with Botox showing degenerative features associated with cancer death (H&E, 400×).