- Title
- The nerve-cancer connection in ovarian cancer
- Creator
- Rodrigues Oliveira, Sónia Marlene
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2018
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Background: Ovarian cancer is usually asymptomatic or the symptoms appear at advanced stages. Despite improvements in treatments, patient survival remains around 40% at 5 years. Ovarian tumours are histologically categorized into serous, mucinous, endometrioid, and clear cell carcinomas. Cervical cancer is the fourth most common female cancer, and affects the cells of the uterine cervix. Squamous cell carcinomas and adenocarcinomas constitute the main histopathological types of cervical cancer and have been strongly linked to specific HPV infections. Progression of tumours is dependent on both the genetic makeup of cancer cells and the surrounding environment - the tumour microenvironment. The tumour microenvironment consists of cells providing structural support, vascular supply, immune cells and a range of growth factors. It has recently been shown that nerve fibres can infiltrate the tumour microenvironment in several solid tumours. Moreover, cholinergic and/or noradrenergic autonomic nerves have been shown to be associated with poor prognosis in prostate, breast and gastric tumours. The role of nerves in gynaecological cancers has not been determined and this encapsulates the main aim of this thesis. Results: First, we investigated nerve infiltration and the expression of nerve growth factor (NGF) in 202 ovarian cancers vs. 18 normal ovarian tissues. Axons were detected by immunohistochemistry using the pan-neuronal marker PGP9.5. Axons could be observed in 72% of normal ovarian tissues but in only 9% of ovarian cancers. Tyrosine hydroxylase, a marker of peripheral sympathetic nerves, was expressed in 89% of normal ovary tissues but only 7% of ovarian tumours. NGF was expressed in 11% of normal ovarian sections and 28% of ovarian cancers. No statistical association was found between the presence of axons, or NGF expression, and age, tumour histological type, grade or lymph node involvement. Next, I used markers for cholinergic (acetylcholine releasing) and a population of sensory nerve fibres (Substance P releasing) to further define the nature of ovarian tumour innervation. Cholinergic innervation was found to be prominent in the ovarian tumour microenvironment while substance P was a rare feature in both normal and ovarian cancer. I also considered the presence of nitrigic axons in the ovarian tumour microenvironment, as detected by the presence of nNOS. Although our nNOS antibody was specific for neurons in the gut many non-neuronal cells were labelled in ovarian cancer making analysis of the nNOS expression difficult. I also investigated the presence of the high affinity NGF receptor, TrkA. While many tumours were TrkA-IR, there was no statistical correlation to clinicopathological parameters or NGF-IR. Instead, other neurotrophins receptors TrkB (BDNF preferred receptor) and p75NTR showed more common expression. Sortilin, receptor for pro-neurotrophins, on the other hand was not detected in the normal ovary and seldom expressed in ovarian tumours. The receptors for the GDNF family – GFRs - were also studied. GFRα1, 2 & 3 were observed and described for the first time in normal ovary and ovarian tumours. All GFRs were detected in both normal and ovarian cancer. GFRα3 showed the most intense IR which may be linked to the antibody characteristics itself. However, all GFRs were detected in blood vessels layers, in varicose across the ovarian stroma and in the GC cells of ovarian follicles. Some ovarian cancer cells were also GFR-IR. In order to further investigate the role of nerves and neurotrophic factor NGF in ovarian cancer in vitro migration and co-culture assays were conducted. My results showed that OVCAR-3 ovarian cancer cells cause 50B11 cells to grown neurites but this is not due to the action of proNGF or NGF. Thus, other neurotrophins as the GDNF family of trophic factors may be major component in the ovarian microenvironment. In vitro Transwell assays with ovarian cancer cells OVCAR-3 showed that acetylcholine has the potential to affect ovarian cancer cells migration, but not NA or SP. Acetylcholine also had a larger effect in ovarian cancer morphology (OVCAR-3 cells numbers, size and shape). Finally, I compared innervation of ovarian cancers to cancers of the cervix. Similarly, to ovarian tumours, I used TMAs of cervical tissue biopsies and screened them with the pan neuronal marker PGP9.5 and neutrophins proNGF and NGF. 93% (272 out of 294 samples) of cervical tumours showed PGP9.5-IR and 87% (257 out of 272) PGP9.5-IR tumours were innervated by axons. No correlation was observed between innervation and clinopathological parameters except for higher PGP9.5-IR in patients under 50 years old. All cervical cancer types were highly innervated (>90% of respective samples). Neuroendocrine cells PGP9.5-immunopositive were also detected in tumours of the cervix. NGF and pro-NGF expressions were increased in cervix tumours. Moreover, we found significant more NGF and proNGF-immunoreactivities in cervix tumours compared with normal cervix. Higher grade tumours also showed more NGF and proNGF expression. Thus, while there was no clear association between axons and neurotrophin expression, the later was significant and may not be required for cervix innervation maintenance or neo-development during cancer progression. Conclusion: Together, these results indicate that nerve infiltration is occasionally found in the tumour microenvironment of ovarian cancer. Cholinergic innervation is predominant over adrenergic nerves. Sensory Substance P axons are a rare feature but ovarian cancer cells express its main receptor, NK1. Moreover, ovarian cancer cells stimulate neurite outgrowth of nociceptive sensory neurons, and cancer cell migration and morphology can be affected by neurotransmitters. Nerve infiltration is a prominent feature of the cervical tumour microenvironment. This is perhaps not surprising given the generally dense innervation of the normal cervix. Moreover, cervix tumours highly expressed neurotrophins NGF and proNGF, particularly in higher grade cancers. Thus, anti-neurotrophin treatments might still be a therapeutic option for cervical cancer patients. Overall the data suggest that the impact of nerves on gynaecological cancers may be different to other solid tumours.
- Subject
- ovarian cancer; tumour microenvironment; Trk receptors; OVCAR-3; 50B11; cervical cancer; axons; TH; cholinergic; peptidergic; NGF; substance P; vAChT; NK1
- Identifier
- http://hdl.handle.net/1959.13/1383870
- Identifier
- uon:31996
- Rights
- Copyright 2018 Sónia Marlene Rodrigues Oliveira
- Language
- eng
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