Myxovirus (influenza virus) resistance A (MxA) is an interferon regulated protein responsible for a specific antiviral state against viral infection. Our lab has previously shown that MxA is up-regulated by androgens in the normal prostate epithelial cells; however, there is no known role for MxA in cancer. Meta-analysis of different expression databases (e.g. NCBI GEO and Oncomine) suggested a strong inverse association between MxA expression and prostate cancer. To confirm these studies, we performed immunohistochemistry on normal prostate and prostate cancer tissues. Our results revealed that MxA expression was indeed decreased in cancerous as compared to normal prostate, indicating that MxA could be transcriptionally down-regulated in cancer. Previous studies indicated that MxA down-regulation could be due to a specific polymorphism in the proximal MxA promoter at position -88. This single nucleotide polymorphism G>T (rs2071430) is involved in modifying the gene expression and interestingly, it harbors an interferon-stimulated response element (ISRE) that is required for expression in response to interferons. The "T" allele restores whereas the "G" allele attenuates ISRE binding, resulting in increased or decreased MxA expression, respectively. Based on these observations we hypothesized that decreased expression of MxA in prostate cancer could be due to the rs2071430 polymorphism. We investigated this polymorphism in genomic DNA from equal number of disease free and prostate cancer samples. The results provide evidence that the GG genotype (low promoter activity) is higher in PCa (72%) as compared to normal (58.6%). The TT genotype (high activity) was higher in normal (5.7%) compared to PCa (2.4%) p

Id proteins are members of basic helix-loop-helix family. However, Id proteins lack the basic binding domain, which prevents DNA binding, and thereby regulates transcription. There are four members in the Id protein family termed Idl-4. In prostate cancer the expression of Idl and Id3 is high, whereas member Id4 expression is low. Decreased expression of Id4 is due to promoter hypermethylation in prostate cancer as well as many other cancers. This observation led us to hypothesize that Id4 acts as a tumor suppressor in prostate cancer. Furthermore, evidence suggests ectopic Id4 expression in metastatic prostate cancer cell line DU145 induced cell cycle arrest, apoptosis, and senescence. In this study, we expanded on these earlier studies to demonstrate that gain of Id4 attenuates cancer phenotype whereas loss of Id4 promotes cancer phenotype in prostate cancer cell lines DU145 and LNCaP, respectively. Upon over-expression of Id4 in DU145 cells (DU145+W4), there was an increase in apoptosis, due to decreased mitochondrial membrane potential (MMP) and increased expression of pro-apoptotic markers (PUMA, BAX, and p21). Inversely, silencing of Id4 in LNCaP cells (LNCaP-Id4) led to decreased apoptosis due to an intact mitochondrial membrane and decrease in the expression of pro-apoptotic markers (PUMA, BAX, and p21). Since BAX, PUMA, and p21 are direct transcriptional targets of p53, these results therefore prompted us to investigate the effect of Id4 on expression and activity of p53. LNCaP cells express wild-type p53. DU145 cells harbor mutant p53 (P223L and V274F), which lies within the DNA binding domain and abrogates p53 transcriptional activity. DU145 cells also express high levels of p53, due extended half-life. Surprisingly, there was decreased expression of p53 in DU145+Id4 cells associated with nuclear localization indicating enhanced transcriptional activity. We investigated p53 DNA binding and transcriptional activity. We determined that mutant p53 in DU145+Id4 cells was transcriptionally active evident by increased luciferase activity and binding of p53 to the promoters of its targets. In LNCaP-Id4, p53 expression was decreased which resulted in decreased p53 transcriptional activity and decreased DNA binding ability. The data suggested that Id4 can restore mutant p53 activity, which is a significant observation. Our results also suggest that Id4 promotes the assembly of a macromolecular complex involving CBP/p300 that results in acetylation of p53 at K373, a critical post-translational modification required for its biological activity. Loss of Id4 in LNCaP cells also abrogated wild type p53 DNA binding and transcriptional activity with concomitant loss of CBP/p300 requirement and decreased acetylation. In conclusion, we demonstrated that loss of Id4 promotes cancer phenotype in LNCaP cells. We also demonstrated that the tumor suppressor activity of Id4 is in part through regulation of CBP/p300 dependent acetylation and function of p53.

Healthy People 2020 was launched in December 2010 with a target human papilloma virus (HPV) vaccination rate of 80%. As of 2014, we were well short of this goal, especially among males, for whom the HPV vaccine became recommended in 2011. An estimated 14-20% of adolescent-aged males had completed the vaccine schedule as of 2015. This is particularly problematic in northeast Tennessee, as multiple risk factors for lower vaccination rates are characteristic of the population, including being white and living in the South. Reasons to decline vaccination vary, and usually involve concerns about safety, efficacy, and necessity. Worries about sexual disinhibition from being vaccinated are often cited by opponents. Parents also perceive a lack of benefit from getting the vaccination starting at age 11 before their sons are sexually active. The media and internet are also barriers to appropriate vaccination in males, as previous research has demonstrated that media coverage is more likely to focus on political controversies instead of benefits, and is more likely to emphasize the benefits to females. Research on improving vaccine uptake on males is currently limited. Doctors who appear knowledgeable and are willing to spend time talking about the HPV vaccine for male patients may increase vaccination rates. Other interventions that may also be effective include vaccinating as part of nurse visits or through school programs. Early studies have been mixed on the effect of patient and parent education on vaccine uptake, although a 2015 review demonstrated that most practice- and community-based educational interventions have some positive effect on uptake. The purpose of this project was to improve HPV vaccine uptake among male patients in a family practice residency program through patient and parent education. After informed consent was obtained, the patients and/or their parents were given a handout produced by the CDC highlighting the benefits of vaccination for males. A chart review was done to determine vaccine coverage among males before the intervention instituted in November of 2016. The intervention was completed in August 2017, and a repeat chart review is currently ongoing to determine vaccine coverage in the post-intervention period. Data collection and analysis is ongoing at the time of abstract submission. We expect a statistically significant increase in the number of male patients who have received any doses of vaccine, and in the number who have completed the vaccine series. Future research should involve broadening the intervention to include local family medicine and pediatrician’s offices to increase vaccine uptake in these populations as well.

Prostate and breast cancer are the leading causes of cancer-related death in men and women, respectively, and metastasis is the primary factor underlying the high mortality rates.1 Snail transcription factor is an important molecule that drives prostate and breast cancer metastasis through the process of epithelial mesenchymal transition (EMT). Proteolytic enzymes that promote invasion and metastasis such as the lysosomal cysteine protease cathepsin L (Cat L) have been shown to degrade E-cadherin, promoting the epithelial mesenchymal transition (EMT).2 It has also been shown that silencing Cat L can inhibit transforming growth factor-beta (TGF-β)-mediated EMT by suppressing Snail transcription factor.3 Several recent studies have highlighted an additional unexpected localization and site of action for Cat L within the nucleus in breast, colon and prostate cancer.4 Natural products have been shown to be efficacious in prevention and possible treatment of cancer.5 Specifically, we have been studying Muscadine Grape Skin Extract (MSKE) as a possible candidate to inhibit Snail signaling. MSKE has previously been shown to promote prostate cancer apoptosis.6 We hypothesized that Snail promotes nuclear localization of Cat L, which promotes EMT associated with increased migration and invasion, and that antagonizing Snail-Cat L signaling would lead to mesenchymal epithelial transition (MET). We showed for the first time that MSKE promotes apoptosis through induction of endoplasmic reticulum stress response and autophagy. Additionally, MSKE could inhibit Snail-mediated EMT via scavenging reactive oxygen species. Moreover, Snail could promote nuclear localization of Cat L, which then promoted cleavage of CDP/Cux, increased Snail transcription and decreased E-cadherin transcription by direct promoter binding of cleaved CDP/Cux, leading to EMT associated with increased migration and invasion. Interestingly, Z-FY-CHO, a small molecule specific inhibitor of Cat L, as well as MSKE could antagonize this signaling by promoting nuclear to cytoplasmic re-localization of Cat L. Therefore, we have dissected novel mechanisms of action of Snail and how it can be antagonized by MSKE natural product.

Transforming growth factor -β (TGF-β) plays an important role in the progression of prostate cancer. It acts as a tumor suppressor in normal epithelial cells but as a tumor promoter in advanced prostate cancer cells. The PI3-kinase pathway has been shown to play integral roles in many cellular processes including cell proliferation, survival, and cell migration in many cell types. PI3-kinase pathway mediates TGF-β effects on prostate cancer cell migration and invasion. Phosphatase and tensin homolog (PTEN), a tumor suppressor gene, inhibits PI3-kinase pathway and is frequently mutated in prostate cancers. In this present study, we investigated possible roles of PTEN in TGF-β effects on proliferation, migration, and the activation of PI3-kinase/AKT pathway in prostate cancer cells. PTEN was expressed in DU145 cells; however PC3 cells lack PTEN expression. TGF-β1 and TGF-β3 had no effect on PTEN mRNA levels but both isoforms increased PTEN protein levels in DU145 and RWPE1 cells, suggesting that TGF-β may mediate regulation of PTEN protein stability. TGF-β1 and TGF-β3 increased PTEN protein levels even in the presence of cycloheximide, a protein synthesis inhibitor, in DU145 cells. In addition, TGF-β upregulated phosphorylation of PTEN, stabilizing PTEN protein. Increase of PTEN protein levels in these cells may also indicate that PTEN may mediate TGF-β effects on cell proliferation. Knockdown of PTEN in DU145 cells resulted in significant increase in cell proliferation which was no longer affected by TGF-β isoforms. PTEN overexpression in PC3 cells inhibited cell proliferation. Knockdown of endogenous PTEN enhanced cell migration in DU145 cells, whereas PTEN overexpression reduced migration in PC3 cells and reduced phosphorylation of AKT in response to TGF-β. Based on these results, we conclude that PTEN plays a role in inhibitory effects of TGF-β on cell proliferation whereas its absence may enhance TGF-β effects on activation of PI3-kinase pathway and cell migration.

Prostate cancer increases its incidence with age after men in their fifth decade as the ratio of estrogen to androgen rises. Epidemiological studies indicated that high levels of estrogens are associated with the high-risk ethnic groups for prostate cancer. Therefore, estrogens may be involved in prostatic carcinogenesis. It is widely believed that the actions of estrogens are mediated by estrogen receptors. However, expression of estrogen receptor in normal prostate and lesions of the gland was controversial. With the recent discovery of second estrogen receptor (ER-β), this issue became more complicated and it needs to be readdressed. In addition, the biological involvement of ER-β in human prostate remains to be investigated. In this study, we demonstrated that human normal prostate epithelial cells express ER-β but not ER-α, suggesting that estrogens act directly on these epithelial cells via ER-β. Using RT-PCR analysis, the transcripts of ER-β were detected in our primary human prostatic epithelial cell cultures that were derived from the ultrasound-guided peripheral zone biopsies and the cells express two estrogen-regulated genes such as progesterone receptor (PR) and pS2. Moreover, we had developed an ER-β antibody with fully characterizations and used it for immunohistochemistry. Results indicated that ER-p protein is expressed in the basal compartment of prostatic epithelium of the gland. Our findings lead to a new hypothesis that estrogens directly act on human prostatic epithelial cells to modulate its biological functions. To investigate expression of ERs in prostate cancer, RT-PCR analysis was used. We found that all three human prostate metastatic cancer cell lines, DU145, PC-3 and LNCaP, express ER-β transcripts while ER-α mRNA expression only in PC-3 cells. Expressions of PR and pS2 in these cell lines are various. LNCaP cells express both PR and pS2 mRNAs but DU145 cells with only PR and PC-3 cells with only pS2. Our immunohistochemical results on prostatic lesions revealed down-regulation of ER-β expression in high-grade of dysplasia and carcinoma of peripheral zone of the prostate compared to their low-grade lesions. This down-regulation in high-grade carcinoma was verified in transcriptional level by RT-PCR analysis on micro dissected normal epithelium and lesion samples of the gland. In the metastasis, ER-β was found to be reactivated as we observed ER-β mRNA expression in prostate cancer cell lines. Recent evidence suggests that ER-β may be antiproliferative factor for a protective effect against the mitogenic activity of estrogens in breast and androgens in prostate. Activation of the receptor may exhibit cell growth inhibition. We demonstrated that antiestrogens [ICI-182,780 (ICI) and 4-hydroxytamoxifen], raloxifene and phytoestrogen (resveratrol), but not estrogens (17β-estradiol and diethylstilbestrol), inhibit growth of DU145 cells which express only ER-β while PC-3 cells with both ERs showed growth inhibition in response to estrogen and antiestrogen treatments. In DU145 cells, the ICI-induced cell growth inhibition was prevented by blockade of ER-β expression using antisense oligonucleotide. It indicated that the inhibition is mediated via ER-p associated pathway. Using flow cytometry, we found that ICI-treatment could induce accumulation of cells at GO-G1 phase of cell cycle. Similarly, this GO-G1 cell accumulation was also induced by raloxifene in DU145 cells. For resveratrol, the treatment exhibited dual effects on cell cycle distribution in DU145 cells. In the early treatment, resveratrol induced cell cycle arrests at GO-G1phase. The prolonged treatment leads to S-phase cell cycle arrest. To study the molecular mechanism of this ER-p associated cell growth inhibition, real-time RT-PCR analysis was used to semi-quantitate the transcript levels of tentative ER-β regulated genes such as telomerase reverse transcriptase (TERT), survivin and thymidylate synthase (TS) in the treated cells compared to those in control. Results demonstrated that the treatment of ICI could down-regulate TERT and survivin mRNA expressions with dose-dependent fashion. As the ICI-treatment, resveratrol downregulated expression levels of TERT, survivin and TS in DU145 cells. Down-regulation of TS may be related to the S-phase cell cycle arrest observed in the prolonged treatment of resveratrol. Taken together, our findings support the concept that ER-β participates in cell cycle regulation in normal and malignant prostatic epithelial cells. Presence of ER-β in basal cells of the prostate acini indicates that the direct actions of estrogens may be involved in the normal physiology of the gland. Loss of this receptor in primary prostate cancer and its re-expression in metastasis suggests the roles of ER-β in the cancer progression. Activation of the receptor by antiestrogen and phytoestrogen induced cell growth inhibition in prostate cancer cells. The mechanism may be mediated by reduction of cell survival factors and eventually decrease in cell viability and induction of cell cycle arrests.

Prostate cancer is the most common noncutaneous cancer among men, yet current diagnostic methods are insufficient and more reliable diagnostic markers need to be developed. The answer that can bridge this gap and enable more efficient diagnoses may lie in microRNAs. These small, single stranded RNA molecules impact protein expression at the translational level and regulate important cellular pathways. Dysregulation of these small RNA molecules can have tumorigenic effects on cells and lead to many types of cancers. Currently the Prostate-Stimulating Antigen (PSA) is used as a diagnostic marker for prostate cancer. However, many factors can elevate PSA levels such as infections and certain medications, consequently leading to false positive diagnoses and unnecessary concern and over treatment with dire outcomes for the patient. Even worse, are the chances of false negative diagnoses, which result in prostate cancer not being diagnosed until its later stages. Therefore, although the use of the PSA level has had its uses in the clinic, it has failed to sufficiently bridge the gap or to distinguish indolent from aggressive disease. It has long been suggested in the literature that microRNAs are drastically altered throughout the course of cancer progression. Here, RNA sequencing was used to identify changes in miR expression profiles diagnostic for prostate cancer patients compared to non-patient controls. The RNA sequencing results were also used to identify normalization miRs to be used as endogenous controls. Confirmatory qRT-PCR was then used to corroborate these results for the top seven dysregulated miRs found from the RNA sequencing data. Data analysis of the Area Under the Curve (AUC) of the Receiver Operating Curves (ROC) of the selected miRs exhibited a better correlation with prostate cancer (AUC Range= 0.819- 0.950) than PSA (AUC of PSA=0.667). In summary, a panel of seven miRs are proposed, many of which have prostate specific targets, which would represent a significant improvement over current testing methods.

One important objective of prostate cancer (PCa) research is to understand the molecular basis underlying the progression of these cancers from an androgen dependent to an androgen independent state. Hypermethylation of the promoter CpG islands is associated with the transcriptional silencing of specific gene sets in each tumor type and subtype. Transcriptional silencing of antitumor genes via CpG island hypermethylation could be a mechanism mediating PCa progression from an androgen-dependent to an androgen-independent state. Hypermethylation associated gene silencing has been reported for a great number of genes in PCa with the exception of the genes that undergo methylation associated silencing specifically during cancer development to androgen independence. The first aim of this thesis is to identify novel glenes which undergo DNA hypermethylation associated gene silencing during the cancer progression. The androgen-dependent (AD, as defined as the inability of celill to proliferate in the absence of androgen) PCa cell line LNCaP gives rise to the androgen-independent (AI) subline LNCaPcs generated by maintaining LNCaP in medium with charcoal-stripped (CS) serum for over 30 passages. This LNCaP cell model was used to identify differentially methylated sequences between the two genomes using the Methylation-Sensitive Restriction Fingerprinting (MSRF) technique. One sequence identified is located in a 5' CpG island, which encompasses part of the promoter, exon 1, and part of intron 1, of the Peroxisomal Membrane Protein 24 KD (PMP24) gene. PMP24 is silenced in concert with the hypermethylation of its CpG island in AI LNCaPcsand PC-3 cell lines. The silencing is reactivated by the treatment with a DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5AZAdC). PMP24 is specifically silenced in PCa cancer cell lines and shows potential antitumor properties. These results demonstrate the utility of MSRF in the identification of novel, differentially methylated DNA sequences in the genome and suggest that hypermethylation-mediated silencing of PMP24 is an epigenetic event involved in PCa progression to androgen independence. The next study investigated the molecular mechanism for DNA methylation associated gene silencing of PMP24 in AI LNCaPcs and PC-3 cell lines. We demonstrated that PMP24 transcription is repressed by the disruption of transcription factor binding to a critical cis-element by hypermethylation of its promoter CpG island. We found a CpG containing activator protein 2 (AP-2) cis-element in the intron 1 of PMP24 whose first CpG dinucleotidle is essential for the sequence-specific protein binding and the promoter activity of the gene. We presented first in cellulo evidence that the methylation of AP-2 cis-element alone but not the whole CpG island, using a newly developed methylated oligonucleotides treatment, is sufficient for the downregulation of PMP24. Our study is the first to report that the silencing mechanism for PMP24 in AI LNCaPcs and PC-3 is mediated by the complete methylation of a single GpG site of AP-2 cis-element in the intron 1 part of the CpG island, which interferes with transcription factor binding. Most interestingly, the promoter CpG island of PMP24 is hypermethylated in AD LNCaP cells with the incomplete methylation specifically at the AP-2 cis-element. The silencing of PMP24 in AD LNCaP cells was reactivated not by the 5AZAdC treatment but by the treatment with Trichostatin A (TSA), a histone deacetylase inhibitor. An alternative silencing mechanism for PMP24 other than the interference with transcription factor binding by methylation is therefore likely involved at this androgen-dependent stage. During the androgen ablation process, this mechanism is either evolved by the spread of methylation in the promoter CpG island or selected against, leading to the methylation-dominant silencing mechanism in the AI cells as seen in LNCaPcsand PC-3 cells. Taken together, this thesis emphasized the important role of DNA methylation in the progression of PCa into androgen independence. Particular respect should be paid to the specific CpG dinucleotides in cis-elements critical for the promoter activity, whose complete methylation could dominate the silencing mechanism which is independent of androgen. This thesis also pointed to the importance of monitoring the effects of cell culture on the methylation status of genes. Most importantly, this thesis raised the possibility that the silencing mechanisms for PMP24 could be different in AD LNCaP cells as compared to AI LNCaPcs and PC-3 cells. Either the evolution of such mechanism or the selectivity against it during the androgen ablation process would result in a methylation-dominant silencing mechanism of the genes such as PMP24 in AI cells and may contribute to the overall androgen independence of the cells.

Prostate cancer, the leading cause of cancer in men, has positive survival rates and constitutes a challenge to men with its side effects. Studies have addressed the bivaritate relationships between prostate cancer treatment side effects masculinity, partner relationship, and quality of life (QOL). However, few studies have highlighted the relationships among prostate cancer treatment side effects (i.e., sexual dysfunction, urinary incontinence), masculinity, and relationship with the partner together on QOL in men. Most studies were conducted with predominately Caucasian sample of men. Miami is a unique multiethnic setting that hosts Cuban, Columbian, Venezuelan, Haitian, other Latin American and Caribbean communities that were not represented in previous literature. The purpose of this study was to examine relative contributions of age, ethnicity, sexual dysfunction, urinary incontinence, masculinity, and perception of the relationship with the partner on the quality of life in men diagnosed with prostate cancer. Data were collected using self administered questionnaires measuring demographic variables, sexual and urinary functioning (UCLA PCI), masculinity (CMNI), partner relationship (DAS), and QOL (SF-36). A total of 117 partnered heterosexual men diagnosed with prostate cancer were recruited from four urology clinics in Miami, Florida. Men were 67.47 (SD = 8.42) years old and identified themselves to be of Hispanic origin (54.3 %, n = 63). Findings demonstrated that there was a significant moderate negative relationship between urinary and sexual functioning of men. There was a significant strong negative association between men’s perceived relationship with partner and masculinity. There was a weak negative relationship between the partner relationship and QOL. Hierarchal multiple regression showed that the partner relationship (β = -.25, t (91) = -2.28, p = .03) significantly contributed overall to QOL. These findings highlight the importance of the relationship satisfaction in the QOL of men with prostate cancer. Nursing interventions to enhance QOL for these men should consider strengthening the relationship and involving the female partner as an active participant.

Background Female stress urinary incontinence (SUI) greatly affects quality of life. The midurethal sling (MUS) procedure has been widely accepted as the standard of care treatment for SUI, although there is little information regarding patients' subjective reports of symptom improvement. Objectives The objective of this study was to identify clinical and demographic characteristics that predict subjective symptom improvement following MUS procedures in women with SUI. Materials and Methods The study design was retrospective cohort. Subjects included women who underwent MUS between 2006 and 2008, returned mailed surveys and met our predefined inclusion criteria. Pre-operative data included demographics, prior surgery, co-morbid diseases, urodynamics and concomitant reconstructive surgery. Subjective improvement was measured by score improvement on the UIQ-7, UDI-6, the UDI stress subscale and Question 3 of the UDI, "Do you experience urine leakage related to physical activity, coughing, or sneezing?" Results The mean age of the study sample was 57 years, parity was 2.5 and BMI was 28. Subjects with lower MUCP demonstrated more improvement on the UIQ-7. ΔUDI-6 stress subscale scores were more sensitive to symptom change than either the ΔUDI-6 or ΔUIQ-7. Older, menopausal subjects with urethral hypermobility and concomitant vaginal suspension showed less improvement than subjects without these characteristics. After controlling for urethral straining angle, PVR, menopause and time out from surgery, older age and concomitant vaginal suspension were associated with persistent post-op symptoms on the UDI-6 Question 3 and age remained the only variable associated with persistent symptoms on the UDI-6 stress subscale. Conclusion Concurrent vaginal suspension and advancing age were risk factors for persistent symptoms following MUS procedures in patients with SUI. Symptoms may recur after 24 post-operative months. Clinicians are encouraged to provide additional preoperative counseling to those women who are at greatest risk for persistent symptoms.