Hypoxia regulated pathways in urological malignancies

Introduction: Kidney Cancer accounts for approximately 2% of all new cancer diagnoses in<br/>the UK each year. Patient survival has improved over the past few decades; however the<br/>mechanisms of this are yet to be fully elucidated. Hypoxia inducible factor isoforms, HIF-1 and<br/>HIF-2, are constitutively expressed in many clear-cell RCCs due to loss of pVHL tumour<br/>suppressor function within the tumour. In vitro, HIF-1 and HIF-2 regulate a differential set of<br/>target genes, although their expression in primary ccRCC clinical samples and their effects on<br/>patient prognosis has yet to be fully understood.<br/><br/>Methods: In this thesis analysis has been performed on all Nephrectomies performed at<br/>Oxford Radcliffe Hospitals for Renal Cell Carcinoma (RCC) from 1983 to 2007. Data extracted<br/>from Charlesworth Research Uro-Oncology Database, CRUD©, provided long-term survival<br/>data, maximal tumour diameter, Fuhrman grade, T-Staging and patient age. A subset of RCCs<br/>from this series (170 consecutive clear cell renal tumours from 1983 to 1999) were analysed<br/>within a tissue microarray and expression of HIF-1 and HIF-2, together with seven primary<br/>target genes (BNIP3, CAIX, CyclinD1, GLUT1, LDH5, Oct-4 and VEGF) was assessed.<br/>Comparison was made with tumour angiogenesis (CD31), tumour stage, Fuhrman grade,<br/>maximum tumour diameter and patient survival. Further work in this thesis analysed a series of<br/>paired VHL (functional and non-functional) ccRCC cell lines, assessing for hypoxic differential<br/>MicroRNA expression.<br/><br/>Results: Analysis of 664 RCCs demonstrated a clear change in kidney cancer specific survival<br/>over the past 24 years, with 5-year survival improving from 42% (1983-1986) to 73% (1999-<br/>2002). The incidence of RCC has increased 10 fold and has a significant association with 4-year<br/>survival. There was no significant change in operative mortality, patient age, Fuhrman grade,<br/>Pathological T-Stage or mean tumour size. However, there was a 5-fold increase in tumours<br/>&lt;6cm, corresponding to an equal fold decrease in tumours 6-8cm, and no change in tumours<br/>&gt;8cm. Tumour size &gt;8cm was a significant prognostic marker. HIF-1 and HIF-2 showed no<br/>correlation and individually, neither HIF-1 nor HIF-2 expression had any prognostic utility;<br/>however a significant time-dependent deterioration of HIF-1 and HIF-2 antigenicity within<br/>paraffin blocks was identified. Angiogenesis (VVI CD31) had a strong negative correlation with<br/>Fuhrman grade and maximal tumour diameter and had prognostic significance, with high levels<br/>associated with good overall survival. Results from microRNA expression arrays found a<br/>specific microRNA (MiR-23a) that was differentially expressed depending upon VHL<br/>functionality and hypoxic conditions. Furthermore microRNA-23a was up-regulated in cells that<br/>expressed both HIF isoforms, and down-regulated in cells that only expressed HIF-2.<br/><br/>Conclusions: Outcome following Nephrectomy for Renal Cell Carcinoma has dramatically<br/>improved over the past 24 years. Increasing incidence and decreasing tumour size at operation<br/>combined with the lack of statistical variation in Fuhrman grade, suggests that earlier detection<br/>of tumours offers subsequent curative treatment by Nephrectomy. Furthermore, stable incidence<br/>rates of tumours &gt;8cm potentially represent alternative tumour biology, which grow rapidly,<br/>avoiding early detection and curative treatment. Although neither HIF isoform nor the seven<br/>HIF target genes was found to influence disease prognosis, the discovery of HIF antigenicity<br/>deterioration with time, is a very important finding and casts into doubt previous literature about<br/>HIF-1 immunostaining in human cancers. The prognostic significance of CD31+ angiogenesis<br/>appears initially counterintuitive, however, CD31+ endothelial cells may represent functional<br/>vessels which protect the tumour from sustained periods of ischaemia, unlike the low VVI<br/>group, from which hypoxia death-resistant clones could arise facilitating tumour metastasis.<br/>This could be very important when considering the effects of biologically targeted antiangiogenic<br/>therapies. Furthermore, the negative correlation with angiogenesis (CD31+) and<br/>Fuhrman grade suggests that vessel functionality and tumour aggressiveness may change with<br/>tumour size. The finding of a specific microRNA that appears to have VHL and HIF dependent<br/>expression extends our understanding of the hypoxic pathway and opens the possibility of<br/>further development of novel targeted therapies.

Name dc:type.qualificationname

Ph.D.

Level dc:type.qualificationlevel

doctoral

Grantor dc:publisher.institution

University of Southampton

Year dc:date.issued

2009

Author dc:creator

• Charlesworth, Philip J.S.

Advisor dc:contributor.advisor

• Johnson, Peter