Background
Epithelial ovarian cancer (EOC) is the leading cause of death from gyneacological malignancy and the fifth most common cause of cancer-related death in women. In 2008 it was estimated that 21,650 new ovarian cancer cases will be diagnosed in the United States and that 15,520 will succumb to the disease [
1]. Despite improvements in surgical techniques and the advent of more targeted therapeutics such as bevacizumab, survival of patients with EOC stands at 45% at five years [
1]. Such poor statistics indicate an urgent requirement to improve our understanding of the molecular mechanisms underlying EOC, which may lead to the development of improved prognostic and predictive assays.
3-hydroxy-3methylglutharyl-coenzyme A reductase (HMG-CoAR) acts as a rate-limiting enzyme in the mevalonate pathway. Although cholesterol represents the main product of this pathway, it also produces a number of non-sterol isoprenoid side products, which have been shown to have a number of tumour-suppressive properties [
2] and to be important regulators of angiogenesis, proliferation, and migration [
3,
4]. HMG-CoAR inhibitors (statins), have demonstrable anti-neoplastic effects
in vitro [
5‐
7] and in xenograft models [
7]. Both the isoprenoid-mediated anti-tumoural properties, and the cholesterol-reducing effects of statins have been suggested to lower the cancer incidence among statin users [
8], although, to date, epidemiological studies have been unable to confirm an association between statin therapy and ovarian cancer risk [
9‐
11].
Members of our group have previously investigated tumour-specific expression of HMG-CoAR by immunohistochemistry (IHC) in 511 incident breast cancer cases within the population-based prospective cohort Malmö Diet and Cancer Study [
12]. This study demonstrated that HMG-CoAR was expressed at various intensities in 82% of the tumours and increased levels of HMG-CoAR protein expression were associated with favourable characteristics, such as a smaller tumour size, low histological grade and estrogen receptor (ER) positivity [
13]. A validation study confirmed these findings and demonstrated that HMG-CoAR was an independent prognostic marker, associated with an improved recurrence free survival (RFS) [
14].
Based on these data, the prognostic power of tumour-specifc HMG-CoAR expression in EOC was examined. This study describes the use of tissue microarray (TMA) technology to investigate the prognostic value of HMG-CoAR in EOC and the use of automated image analysis to quantify HMG-CoAR expression.
Discussion
This is, to our knowledge, the first study to describe tumour-specific HMG-CoAR expression in EOC. Cytoplasmic expression of HMG-CoAR was evident in varying intensities in 65% of the tumours. Although HMG-CoAR was not associated with disease stage, grade, estrogen receptor or Ki-67 expression, it was associated with a prolonged RFS. Manual and automated quantification of HMG-CoAR expression were both associated with a prolonged RFS and Cox multivariate proportional hazards analysis confirmed that this was independent of stage and grade. These findings support previous results from our group describing the association between tumour-specific HMG-CoAR expression in breast cancer and a less aggressive tumour phenotype [
13,
14].
As HMG-CoAR is the rate-limiting enzyme of the mevalonate pathway, these data add further evidence of this pathway's importance in tumour development and progression. While HMG-CoAR inhibitors, also known as statins, have demonstrated excellent efficacy in the treatment of hypercholesterolemia and cardiovascular disease, their role in oncology remains relatively unproven. Despite an ever-growing body of literature describing the anti-neoplastic properties of statins, epidemiologic data regarding their preventive effect against cancer in general, and EOC in particular, remain inconclusive [
9,
10,
18‐
22]. A recent pre-operative window trial of ductal carcinoma in situ and stage one breast cancer was the first to demonstrate that statins can inhibit proliferation and increase apoptosis
in vivo [
23]. This raises the possibility that the combination of statins and well-established chemotherapeutic agents may be an option in the neo-adjuvant setting in other tumour types also.
HMG-CoAR activity in tumour cells is elevated and dysregulated. HMG-CoAR activity in leukemia cells [
24,
25] and lung carcinoma cells [
26] are 3-8-fold and 2-fold higher, respectively, than in normal cells. Furthermore, statin induced mevalonate depletion has been shown to result in an adaptive induction of HMG-CoAR expression in chinese hamster ovary cells [
27] and MCF-7 breast cancer cells [
28]. Treatment of MCF-7 cells with mevastatin resulted in a 10- to 15-fold induction of HMG-CoAR activity in association with a 2.5- to 3.5-fold induction of HMG-CoA reductase mRNA expression [
28], suggesting that treatment with statins may increase tumour specific HMG-CoAR expression
in vivo, however this remains to be fully elucidated. It seems counterintuitive that statins cause an increase in tumour-specifc HMG-CoAR expression however this is felt to be secondary to a loss of sterol mediated inhibition of HMG-CoAR transcription in tumour cells [
2]. The statin induced increase in HMG-CoAR results in an increase non-sterol isoprenoid side products, with their associated tumour-suppressive properties, which may explain the efficacy of statin in treating tumour cells
in vitro [
2]
Kato
et al recently demonstrated that lypophillic statins induce apoptosis in ovarian cancer cells, and also postulated that HMG-CoAR expression predicted response to statin treatment [
29]. In vitro data demonstrate that statins induce apoptosis and inhibit tumour formation in soft agar in ovarian cancer cells via activation of the JNK pathway and pro-apoptotic proteins such as Bim [
30]. Additionally statin induced suppression of RhoA has been shown to inhibit peritoneal dissemination of ovarian cancer cells
in vivo [
31]. Likewise high-dose lovastatin has been shown to inhibit tumour proliferation in a xenograft model of anaplastic thyroid cancer [
32].
It has been postulated that the anti-neoplastic effects of statins could be attributed to their ability to increase HMG-CoAR activity in tumour cells, thus leading to the production of non-sterol bi-products of the mevalonate pathway [
2]. Increased HMG-CoAR activity increases the synthesis of farnesyl diphosphate and geranylgeranyl diphosphate. These substrates provide the isoprenoid moieties for the post-translational modification of the cysteine residue of the conserved carboxyl terminus sequence of diverse proteins - known as prenylation [
2]. Prenylation has been shown to have a number of tumour suppressive actions including the induction of apoptosis [
33], the initiation of G1 arrest [
33] and the suppression of small G-protein receptors and intracellular growth pathways [
34]. HMG-CoAR expression could be a surrogate marker of protein prenylation, thus explaining our findings that increased levels of HMG-CoaR are associated with an improved prognosis in both breast and EOC.
Conclusion
In summary, this is the first description of tumour-specific HMG-CoAR expression in EOC. Given that all of the patients in this study received adjuvant platinum-based chemotherapy, these data suggest that the addition of statins to traditional chemotherapeutic regimens may be an efficacious and well-tolerated strategy in EOC. Although data were not available on statin use in this cohort, a growing body of experimental evidence exists describing a synergism between cisplatinum and statins in vitro [
35‐
37]. Recent
in vivo data confirmed that statins have an anti-neoplastic effect in breast cancer [
23] and it is anticipated that ongoing prospective trials will shed more light on this issue [
38]. It should also be noted that while further studies are required to investigate the value of HMG-CoAR expression as a predictive marker of response to statin treatment, our results provide evidence to justify prospective randomized controlled trials examining the addition of statins to standard adjuvant chemotherapeutic regimens for EOC.
DJB, JB, MF and CO'H are practising obstetricians and gynaecologists, DJB, ER, DO'C, WG and MU are research scientists with a special interest in automated quantification of immunohistochemistry, FP and KJ are practising pathologists.
Acknowledgements
The authors thank Elise Nilsson for excellent technical assistance. This study was supported by grants from the Health Research Board of Ireland, the Knut and Alice Wallenberg Foundation, the Swedish Cancer Society, Gunnar Nilsson's Cancer Foundation and the research funds of Malmö University Hospital. The UCD Conway Institute is funded by the Programme for Third Level Institutions (PRTLI), as administered by the Higher Education Authority (HEA) of Ireland.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
DJB designed the study, performed image analysis, statistical analysis and drafted the manuscript, JB performed manual analysis of IHC and statistical analysis, ER performed image analysis, MF developed the patient database, FP designed the study and helped draft the manuscript, MU designed the study and helped draft the manuscript, WG provided image analysis platforms and designed the study, DO'C designed the study and drafted the manuscript, CO'H provided TMAs and designed the study, KJ performed IHC, manual analysis and drafted the manuscript. All authors read and approved the final manuscript.