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Denise S. O'Keefe, PhD

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The most common molecular changes seen in prostate cancer are epigenetic (non-mutational) in nature, such as the hypermethylation of gene promoter sequences leading to transcriptional repression.   In fact, more than 90% of prostate cancers exhibit alterations in the DNA structure of particular genes, resulting in the “switching-off” of a number of tumor-suppressor type genes. In addition, there are other epigenetic changes frequently seen in prostate tumors that allow expression of the so called “junk DNA” in our genome, which causes disruption of the nuclear structure and subsequent ectopic expression of these “retrotransposons”, likely resulting in ectopic expression of growth-promoting genes, and repression of growth regulating genes. Our lab investigates the potential mechanisms leading to these epigenetic changes to better understand the etiology of prostate cancer and thus develop novel tools for diagnosis, prognosis and treatment of the disease.

Diet and Disease: The role of folate in the initiation and progression of Prostate Cancer. 
Many factors can reportedly alter the epigenetic status of DNA; however, folic acid is of notable importance.  Folate, as part of the one carbon cycle, is critical for the synthesis of S –adenosyl methionine, which contributes the methyl group for methylation reactions.  In 1998 the U.S. government mandated fortification of the food supply with folic acid, resulting in a tripling of the mean serum folate level in the U.S. population.  Recent studies indicate that dietary folate may affect epigenetic programming, even in adults.  It also seems that the epigenetic changes that occur differ, depending on the tissue type and the time at which dietary manipulation occurs.  The prostate has a particularly high requirement for folate and seems especially susceptible to alterations in DNA methylation.  Moreover, epidemiological evidence suggests that higher plasma folate levels increase the rate of prostate cancer.  These findings have led us to study the effects of dietary folate on prostate cancer.  Indeed, we have evidence that two proteins intimately involved in intracellular folate regulation contribute to prostate cancer.  PSMA, a unique folate hydrolase that may increase intracellular folate levels, can induce invasive prostate cancer in a mouse model.  At the same time, we have identified a folate exporter that is down-regulated in prostate tumors, and undergoes epigenetic silencing in prostate cancer cell lines.  Interestingly, this down-regulation occurs under conditions of folate deprivation in an in vitro model of prostate cancer.  Currently, we are evaluating global and specific epigenetic changes in primary human prostate tissue under different conditions of dietary folate.

In our most recent study we found that amongst 50 patients with Gleason Grade 7 tumors, there was more than a 100-fold variation in fasting serum folate levels. While folate contributes to all biologic methylation reactions, it is also necessary for nucleotide synthesis and tumor cells grown in vitro proliferate directly in response to available folate.  When we ranked our patients based on their serum folate levels, we found that those in the top quintile for serum folate had tumors that proliferated, on average, 6 times more quickly than those in the lowest quintile for serum folate.  These results are consistent with the findings of other clinical studies where there was a significant increase in patients diagnosed with prostate cancer in groups supplemented with levels of folic acid equivalent to 2 and one-half multivitamins, on a daily basis.  We are currently studying the impact of dietary folate on human prostate cancer, and in future plan to expand these studies to other cancer sites, dependent on available funding.    Grant Support:  Wendy Will Case Cancer Fund, AICR, NIH

Complementary Therapy for Androgen Independent Prostate Cancer:  

The inability to adequately treat androgen-independent prostate cancer causes the majority of patient deaths, prompting us to explore new therapeutic strategies. The androgen dihydrotestosterone binds to the androgen receptor (AR), inducing a homodimer formation that activates expression of genes with an androgen response element in their promoter.  As androgen stimulates prostate cell growth and inhibits glandular epithelial cell death, androgen ablation therapy decreases tumor size. However, the vast majority of patients eventually develop androgen-independent tumors.  At this point, the standard of care is treatment with the microtubule inhibitor, Docetaxel.  Unfortunately, the tumors generally become docetaxel-resistant, probably because they alter gene expression so as to pump the drug out of the cell.  We have identified a multidrug transporter, ABCG2, that is normally under-expressed in prostate tumors, but rapidly upregulated in response to docetaxel treatment, rendering the cells docetaxel-resistant.  If we could inhibit ABCG2 function during docetaxel treatment, we may be able to block docetaxel resistance.  ABCG2 inhibitors exist, but have never been used to treat prostate cancer.  Furthermore, some dietary components, including resveratrol (found in red wine, some nuts and grapes) and curcumin (found in the yellow curry spice, turmeric), are thought to be naturally potent inhibitors of ABCG2 function.  We are investigating the role of these dietary components with regards to docetaxel resistance, in the hope that patients could supplement either one or both of these to their diet, and extend the time to recurrence after docetaxel therapy, or combat the tumor altogether.  Support: ABCG2 and docetaxel resistance funded by a DOD predoctoral fellowship (Katy Sobek), dietary studies currently not supported.

Field Defects and Prostate Carcinogenesis:

A field defect describes an area of histologically normal tissue that contains epigenetic or mutational defects also seen in tumors from adjacent tissue.  Thus, these changes likely represent some of the earliest events in carcinogenesis.  The silenced genes marking the field defect may make ideal therapeutic targets since it is theoretically possible to use drugs to reverse epigenetic changes and switch the genes back on.  Mining microarray data, we have identified several genes with decreased expression in prostate cancer, for example the gene that encodes the pro-apoptotic protein ASC.  Real-time PCR demonstrated the down-regulation of these genes in prostate cancer cell lines, but not in normal prostate lines.  This decrease in expression resulted from promoter methylation.  Using methylation specific PCR on microdissected patient specimens, we showed that methylation occurred not only in tumors, but also in high-grade prostatic intraepithelial neoplasia and normal tissue adjacent to the tumor.  Importantly, we did not detect methylation in prostate tissue from cancer-free donors.  These observations suggest that our genes act as markers for a field defect.  Furthermore, we have found that the most common field-defect is marked by hypomethylation of LINE-1 retrotransposon sequences.  Retrotransposons are normally heavily methylated as their expression can lead to genomic disruption and ectopic expression or repression of nearby genes.  However we have shown that these so called “junk DNA” sequences are actually activated not only as part of the field-defect, but also in frank prostate cancer.  In addition, we have also shown that expression of these ancient retroviral-like sequences corresponds with activation of immune response pathways normally reserved for response to viral infection, and perhaps related to the frequent inflammation seen in prostate tumors and prostate tissue, that has often been implicated as a potential etiologic agent for this disease. Grant Support:  Edwin Beer Award, D.O.D., NIH

Methylation Profiling as a Clinical Tool in Prostate Cancer:

Our lab is also working toward establishing prostate cancer methylation profiles, patterns of methylated and inactivated genes, to predict the clinical behavior of  particular tumors.  This capability would allow physicians to tailor treatment specifically to each patient.  Methylation profiling, already proven useful in the management of other cancers, has particular relevance to prostate cancer. Based on clinical T stage, biopsy Gleason score and preoperative serum PSA, prostate cancer patients are stratified into three risk groups for recurrence following radical prostatectomy: low, intermediate and high.  These criteria, however, lack prognostic accuracy for the intermediate group, underscoring the need for new markers.   Another important problem in prostate cancer is the management of patients with high-grade prostatic intraepithelial neoplasia (HGPIN), a putative precursor to prostate cancer.  The presence of HGPIN on a needle biopsy often, but not always, indicates the presence of cancer within the prostate, necessitating additional biopsies.  Furthermore, the cancer might not be adjacent to or even on the same side of the prostate that contains the HGPIN. Thus, methylation profiles of HGPIN that could accurately predict the presence of cancer would be very beneficial.  We can measure, relatively inexpensively, promoter methylation on both biopsy and paraffin-fixed archived samples. Hence, we are fortunate that, through Urology, we have access to one of the largest and most comprehensive prostate tissue banks in the U.S., allowing us to thoroughly test our hypotheses and develop accurate approaches toward methylation profiling. Grant Support:  Edwin Beer Award and D.O.D.

Identification of a novel retrovirus in Benign Prostatic Hyperplasia (BPH)

BPH describes a benign condition experienced by most men as they age, and is a result of increased proliferation (growth) of fibroblast and epithelial cells surrounding the urethra, which over time can form “nodules” and result in compression of the urethra and subsequently obstruction of urinary flow. Symptoms of BPH include urinary hesitancy, frequent urination, dysuria (painful urination), increased risk of urinary tract infections, and urinary retention.  Treatment includes surgery and pharmacologic options, although the cause of BPH is unknown.  Using tissue from patients who have undergone surgical treatment to alleviate BPH, we discovered that the affected tissue appears to have altered gene expression patterns when compared to normal prostate tissue.  This is not unexpected, as the tissue is clearly growing more quickly.  However we identified what appears to be increased expression of genes related to an antiviral response.  Given the recent findings of a novel virus, XMRV, found in some prostate tumors, we analyzed the BPH affected tissue for viral infection.  We found that the majority of tissue from symptomatic BPH patients contained low levels of a virus not previously found in humans. Sequencing confirmed that the virus consists of 2 variants, is not XMRV, and likely produces a protein that has been related to inflammation in other species.  Interestingly, the exact virus sequence differs among patients, suggesting that upon infection of the tissue, the virus undergoes replication.  Furthermore, sequencing revealed that the virus is likely transcriptionally regulated by androgens, which is consistent with the fact that cell growth in BPH is androgen-dependent and the classic non surgical treatment for BPH is inhibition of DHT (an androgen) production.  Both variants of the virus have submitted to the USPTO as a provisional patent, as they may be a therapeutic target for this disease.  In addition, we are currently seeking funding to assist in the development of a vaccine, which could potentially eradicate BPH if this novel virus causes the disease, or even if it is just expressed (as a “bystander”) in BPH-affected tissue.