Hypoxia regulated pathways in urological malignancies

Abstract

Introduction: Kidney Cancer accounts for approximately 2% of all new cancer diagnoses in
the UK each year. Patient survival has improved over the past few decades; however the
mechanisms of this are yet to be fully elucidated. Hypoxia inducible factor isoforms, HIF-1 and
HIF-2, are constitutively expressed in many clear-cell RCCs due to loss of pVHL tumour
suppressor function within the tumour. In vitro, HIF-1 and HIF-2 regulate a differential set of
target genes, although their expression in primary ccRCC clinical samples and their effects on
patient prognosis has yet to be fully understood.

Methods: In this thesis analysis has been performed on all Nephrectomies performed at
Oxford Radcliffe Hospitals for Renal Cell Carcinoma (RCC) from 1983 to 2007. Data extracted
from Charlesworth Research Uro-Oncology Database, CRUD©, provided long-term survival
data, maximal tumour diameter, Fuhrman grade, T-Staging and patient age. A subset of RCCs
from this series (170 consecutive clear cell renal tumours from 1983 to 1999) were analysed
within a tissue microarray and expression of HIF-1 and HIF-2, together with seven primary
target genes (BNIP3, CAIX, CyclinD1, GLUT1, LDH5, Oct-4 and VEGF) was assessed.
Comparison was made with tumour angiogenesis (CD31), tumour stage, Fuhrman grade,
maximum tumour diameter and patient survival. Further work in this thesis analysed a series of
paired VHL (functional and non-functional) ccRCC cell lines, assessing for hypoxic differential
MicroRNA expression.

Results: Analysis of 664 RCCs demonstrated a clear change in kidney cancer specific survival
over the past 24 years, with 5-year survival improving from 42% (1983-1986) to 73% (1999-
2002). The incidence of RCC has increased 10 fold and has a significant association with 4-year
survival. There was no significant change in operative mortality, patient age, Fuhrman grade,
Pathological T-Stage or mean tumour size. However, there was a 5-fold increase in tumours
<6cm, corresponding to an equal fold decrease in tumours 6-8cm, and no change in tumours
>8cm. Tumour size >8cm was a significant prognostic marker. HIF-1 and HIF-2 showed no
correlation and individually, neither HIF-1 nor HIF-2 expression had any prognostic utility;
however a significant time-dependent deterioration of HIF-1 and HIF-2 antigenicity within
paraffin blocks was identified. Angiogenesis (VVI CD31) had a strong negative correlation with
Fuhrman grade and maximal tumour diameter and had prognostic significance, with high levels
associated with good overall survival. Results from microRNA expression arrays found a
specific microRNA (MiR-23a) that was differentially expressed depending upon VHL
functionality and hypoxic conditions. Furthermore microRNA-23a was up-regulated in cells that
expressed both HIF isoforms, and down-regulated in cells that only expressed HIF-2.

Conclusions: Outcome following Nephrectomy for Renal Cell Carcinoma has dramatically
improved over the past 24 years. Increasing incidence and decreasing tumour size at operation
combined with the lack of statistical variation in Fuhrman grade, suggests that earlier detection
of tumours offers subsequent curative treatment by Nephrectomy. Furthermore, stable incidence
rates of tumours >8cm potentially represent alternative tumour biology, which grow rapidly,
avoiding early detection and curative treatment. Although neither HIF isoform nor the seven
HIF target genes was found to influence disease prognosis, the discovery of HIF antigenicity
deterioration with time, is a very important finding and casts into doubt previous literature about
HIF-1 immunostaining in human cancers. The prognostic significance of CD31+ angiogenesis
appears initially counterintuitive, however, CD31+ endothelial cells may represent functional
vessels which protect the tumour from sustained periods of ischaemia, unlike the low VVI
group, from which hypoxia death-resistant clones could arise facilitating tumour metastasis.
This could be very important when considering the effects of biologically targeted antiangiogenic
therapies. Furthermore, the negative correlation with angiogenesis (CD31+) and
Fuhrman grade suggests that vessel functionality and tumour aggressiveness may change with
tumour size. The finding of a specific microRNA that appears to have VHL and HIF dependent
expression extends our understanding of the hypoxic pathway and opens the possibility of
further development of novel targeted therapies.

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Identifiers

ORCID for Peter Johnson: orcid.org/0000-0003-2306-4974

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