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Martin Tenniswood, Ph.D.
The Role of Vitamin D in Prostate Cancer Progression
In collaboration with Professor JoEllen Welsh (Graduate Student: Wei-Lin Winnie Wang)
In November 2011, the U.S. Preventive Services Task Force recently recommended against PSA screening for prostate cancer in healthy men, because of the "moderate to high certainty that the service has no benefit and that the harms actually outweigh the benefits". While the task force acknowledges that the test is sensitive and accurate with a low false positive rate, they contend that "there is little evidence PSA testing saves lives, but rather that many men instead suffer from impotence, incontinence, heart attacks related to treatment of tiny tumors that would never kill them". Not surprisingly, many urologists disagree, and a number of prominent urologic oncologists have recently published a strongly dissenting opinion. At the center of this controversy is the fact that only 3 out of 10 men diagnosed with prostate cancer on the basis of PSA testing and needle biopsy actually need definitive therapy, while 7 out 10 men have indolent disease and will never need treatment. However, in the absence of a test that accurately distinguishes indolent (non-lethal) and aggressive (lethal) disease, the vast majority of most men diagnosed with the prostate cancer are being treated as though they have aggressive prostate cancer. In addition to the morbidities noted above, this costs the healthcare system an estimated $2.6B per year in unnecessary treatment costs. Thus the problem is not with the PSA test itself, but with the current inability to distinguish between indolent and aggressive disease once the tumor has been confirmed by needle biopsy.
Epidemiological evidence suggests that maintaining adequate serum vitamin D3 (D3) levels is important in preventing prostate cancer and in a recent study, more than 75% of men diagnosed with prostate cancer are D3 deficient. D3 modulates transcription after binding to the vitamin D receptor (VDR) which heterodimerizes with retinoid X receptor (RXR) and engages vitamin D response elements (VDREs) to regulate the transcription of target genes. While prostate cancer is generally thought of as an androgen-dependent disease, it is paradoxical that the age dependent incidence of the disease (and associated mortality) increases after serum testosterone (T) levels have decreased significantly. The incidence of prostate cancer first increases between 55-64 years of age, when the average serum testosterone levels drop below 20 nm/L and free testosterone has decreased by 50%.
At the time of diagnosis the majority of men have declining serum T and low D3 levels, leading to the hypothesis that the two hormones interact to block prostate tumor initiation and/or progression, and D3 may play a much more prominent role in blocking tumor progression than previously realized. Our investigation of the molecular mechanisms underlying this phenomenon using miRNA and mRNA genome wide expression studies in two androgen receptor(AR) positive cell lines(LNCaP and PC-346C) has established that a cohort of T and D3-modulated miRNAs regulate the expression of critical genes responsible for regulation of PCa tumor progression. In particular, in LNCaP cells, T and D3 have been shown to significantly induce the expression of a small cohort of miRNAs and to synergistically repress the expression of the miR17/92 (Oncomir-1) cluster. Upregulation of the latter cluster has been implicated in the progression of several solid tumors through the modulation of E2F family members and cyclin D1. This suggests that the loss of T- and D3-mediated suppression of this cluster plays an important role in the progression of prostate cancer. Validated targets of these miRNAs in the prostate, at the mRNA and protein level include c-Myc, cyclin D1 and members of the E2F family of transcription factors. Elevation of E2F transcription factors, and the disruption of the transcriptional activity of cyclin D1 have been implicated in the disruption of androgen receptor signaling and the failure of advanced tumors to respond to bicalutamide therapy, one of the major biochemical hallmarks of castration resistant prostate cancer (CRPC).
We are currently investigating the hypothesis that dysregulation of T- and D3-regulated miRNAs as a result of declining T and D3 levels in aging men precipitates prostate cancer progression (Figure 1). These data have two implications:
- miRNA profiles will reflect the biochemical status of tumor cells, and can be used to distinguish indolent versus aggressive tumors.
- Maintenance of adequate levels of serum D3 and T may significantly slow, or reverse, the progression of prostate cancer.
Targeting Acetylation in Triple Negative Breast Cancer: a Multi-omics Approach
(Graduate Students: Namita Chatterjee and Ann-Christin Gaupel)
According to the Surveillance, Epidemiology and End Results (SEER) database the survival rate varies very significantly and depends largely on the sub-type of breast cancer. Women diagnosed with tumors confined to the primary site that are classified as luminal A (approximately 60-70% of the cases in North America) have a relative five year survival rate of 95-98%. However women diagnosed with regional invasive disease or distant metastases that are classified as triple negative breast cancer (TNBC) (4% of cases), or who present with inflammatory breast cancer (IBC) (2% of cases), have a five year survival rate of less than 25%, and despite their relatively low incidence represent nearly 20% of all breast cancer related deaths. Both diseases, but particularly IBC, disproportionately afflict underserved Afro-American and Hispanic women. It is now evident that the genetic and epigenetic changes in advanced stage tumors make them more difficult to treat successfully. While anti-estrogens and aromatase inhibitors have improved the survival for a significant population of women with estrogen receptor positive (ER+) tumors, and Herceptin (Trastuzumab) has improved the survival of many women with tumors that over-express the HER2 receptor (ERRB2). Most advanced tumors do not respond to these therapies, and many women afflicted with TNBC and IBC succumb to their disease, despite very aggressive therapies. Clearly there is a need for new therapies for breast cancer, and refinement of current therapies to improve their effectiveness, particularly for triple negative and inflammatory breast cancers.
Recent in vitro studies from our laboratory has shown that IBC cell lines are extremely very sensitive to the type 1 histone deacetylase (HDAC) inhibitors, trichostatin A (TSA) and CG-1521. In arrayed based analysis CG-1521 modulates the expression of a small number of miRNA that target the very specific gene ontologies associated with spindle assembly, furrow formation and abscission, resulting in mitotic catastrophe and cell death.
In addition to altering the acetylation of histones HDAC inhibitors alter the acetylation of a number of transcription factors including p53 and ER (Roy & Tenniswood, 2007), influencing nuclear retention, protein binding partners and DNA binding activity of these transcription factors. The alteration in activity is predominantly due to changes in the charged surfaces associated with the bromodomains responsible for the protein-protein associations required for the formation of active transcription factor complexes. It has also become apparent that the lysine residues in many transcription factors that are acetylated by HATs are also targets for E3-ligases that ubiquitinate them, targeting the protein for proteasomal degradation. Thus the acetylation state of these transcription factors influences their steady state protein level and their activity. This has led to the hypothesis that transcription factors exist as “meta-stable” networks that are modulated through the balance between acetylation and ubiquitination (Fig. 1), which is determined by the relative activity of the four classes of enzymes that modulate the post-translational modification of the targeted lysines. While the bulk of the acetylation/deacetylation activity in the cell is directed toward histones, acetylation and deacetylation cycles of non-histone proteins have a major biological impact on the responsiveness of tumors to current therapeutics, and targeted disruption of one or more of the enzymes will have significant consequences on transcriptional processes.
Identification of these key acetylation sites in IBC cell lines and tumors will provide new targets for therapeutic intervention. Recent studies from our laboratory and others suggest that hydroxamic acid-based HDAC inhibitors are potent inhibitors of cancer cell growth that transcend the breast cancer subtypes, abrogating cell growth by arresting cell cycle and inducing apoptosis in luminal, TNBC and IBC cell lines. Our data confirm that acetylation modulates cell signaling at multiple levels.
Given the paucity of therapeutic targets for IBC and TNBC we have decided to take a global genomic, proteomic and acetylomic approach., using a combination of high content analyses (HCA) and high throughput screening (HTS) to identify common targets for new therapeutic invention. We are evaluating the effects of current therapies on the balance between acetylated and non-acetylated transcription factors to develop a rationale for combination therapy to synergistically induce cell death and thus allow lower doses of cytotoxic drugs needed to produce remission or cures. Our overall goal is to globally access the sensitivity of each cell line to selected HDAC inhibitors, and correlate the acetylation status of non-histone proteins (transcription factors and cytoplasmic proteins) with drug sensitivity. We will also assess the effects of co-administration of one or more HDAC inhibitors on the responsiveness of the cell lines to paclitaxel or radiation in orthotopic xenograft models.