Evidence That Selenium Binding Protein 1 Is a Tumor Suppressor in Prostate Cancer

<div><p>Selenium-Binding Protein 1 (SBP1, SELENBP1, hSP56) is a selenium-associated protein shown to be at lower levels in tumors, and its lower levels are frequently predictive of a poor clinical outcome. Distinguishing indolent from aggressive prostate cancer is a major challenge in disease management. Associations between SBP1 levels, tumor grade, and disease recurrence following prostatectomy were investigated by duplex immunofluorescence imaging using a tissue microarray containing tissue from 202 prostate cancer patients who experienced biochemical (PSA) recurrence after prostatectomy and 202 matched control patients whose cancer did not recur. Samples were matched by age, ethnicity, pathological stage and Gleason grade, and images were quantified using the Vectra multispectral imaging system. Fluorescent labels were targeted for SBP1 and cytokeratins 8/18 to restrict scoring to tumor cells, and cell-by-cell quantification of SBP1 in the nucleus and cytoplasm was performed. Nuclear SBP1 levels and the nuclear to cytoplasm ratio were inversely associated with tumor grade using linear regression analysis. Following classification of samples into quartiles based on the SBP1 levels among controls, tumors in the lowest quartile were more than twice as likely to recur compared to those in any other quartile. Inducible ectopic SBP1 expression reduced the ability of HCT-116 human tumor cells to grow in soft agar, a measure of transformation, without affecting proliferation. Cells expressing SBP1 also demonstrated a robust induction in the phosphorylation of the p53 tumor suppressor at serine 15. These data indicate that loss of SBP1 may play an independent contributing role in prostate cancer progression and its levels might be useful in distinguishing indolent from aggressive disease.</p></div

SBP1 decreases soft agar colony formation in HCT116 cells.

<p>The ability of HCT116 cells with and without SBP1 to form colonies in soft agar was measured. Cells induced to express SBP1 as well as SBP1-null cells were mixed with media containing 0.4% agarose, and each group was plated in triplicate on 6 well plates coated with 0.6% agarose in media. Each well contained 5x10⁴ cells, and colonies larger than 0.5mm were counted on day 15 of growth (A). Error bars represent S.D. (*p<0.05 n = 3).</p

SBP1 levels differ in human prostate tissue.

<p>An example of the image of a core obtained from the outcome CPCTR tissue microarray stained with anti-SBP1 (red) antibodies followed by Alexa488 goat anti-rabbit and Alexa647 goat anti-mouse respectively. Nuclei were counterstained with DAPI (blue). A is an example of nuclear localized SBP1, B shows cytoplasmic SBP1, and C is an example of very low SBP1 expression in prostate tissue.</p

Patients whose tissue was in the lowest quartile of nuclear SBP1 are significantly more likely to recur after radical prostatectomy than patients whose tissues had higher nuclear SBP1.

<p>Odds ratios (OR) and 95% confidence intervals (CI) for prostate cancer recurrence by quartile of SBP1 expression. Expression levels were measured using the VECTRA quantitative imaging system, and all OR estimates are adjusted for PSA, Gleason grade, tumor stage, and patient age at diagnosis.</p><p>Patients whose tissue was in the lowest quartile of nuclear SBP1 are significantly more likely to recur after radical prostatectomy than patients whose tissues had higher nuclear SBP1.</p

Lower SBP1 levels in the nucleus are significantly associated with advanced prostate cancer.

<p>Patients in the prostate cancer outcome tissue microarray were distributed into three categories (Gleason Category) based on Gleason score (A). Mean quantified tissue SBP1 was ranked for all patients and assigned a score from 1–368 based on relative SBP1 levels where the patient with the lowest tissue SBP1 received a score of 1, and the patient with the highest SBP1 levels received a score of 368. Box-and-whisker plots indicate the range of scores from patients in each category (vertical lines) and the range of the first and third quartile in each category (boxes). The horizontal line inside the box indicates the median. The association between SBP1 and Gleason Category was determined using SBP1 in the nucleus (B), cytoplasm (C) and total cell (D).</p

SBP1 does not change the proliferation of HCT116 cells.

<p>HCT116-TetSBP1 cells with (+Dox) and without (-Dox) SBP1 were grown for 6 days on a 96 well plate. Cells were treated with 1ug/mL doxycycline 48 hours before the 0 hour time point. Five hundred cells were seeded in triplicate 24 hours before the 0 hour time point. Fluorescently labeled dsDNA from each well of a 96 well plate was quantified at four time points- 0, 48, 96, and 144 hours, and error bars represent standard deviations at each time point.</p

SBP1 sensitizes cells to treatment with colon cancer chemotherapeutic 5-FU.

<p>The growth of HCT116-TetSBP1 cells was measured after a 6 day treatment with 5uM 5-FU. Double stranded DNA was measured as an indicator of cell growth at four time points- 0, 48, 96, and 144 hours, and error bars represent standard deviation at each time point (*p<0.05, **p < 0.01). Five hundred cells were seeded in triplicate 24 hours before 0 hour time point, and fluorescently labeled dsDNA from each well of a 96 well plate was quantified after cells were lysed while still attached to the plate.</p

There was no association between the nuclear to cytoplasmic ratio of SBP1 and prostate cancer recurrence.

<p>Odds ratios (OR) and 95% confidence intervals (CI) for prostate cancer recurrence by quartile of nuclear to cytoplasmic SBP1 expression. Expression level was measured by the VECTRA quantitative imaging system, and all OR estimates are adjusted for PSA, Gleason grade, tumor stage, and patient age at diagnosis.</p><p>There was no association between the nuclear to cytoplasmic ratio of SBP1 and prostate cancer recurrence.</p

Lower nucleus:cytoplasm SBP1 ratio is significantly associated with advanced prostate cancer.

<p>Patients in the prostate cancer outcome microarray were distributed into three categories (Gleason Category) based on Gleason score (A). Mean quantified tissue SBP1 was ranked for all patients and assigned a score from 1–368 based on relative SBP1 levels where the patient with the lowest tissue nuclear:cytoplasmic ratio of SBP1 received a score of 1, and the patient with the highest ratio received a score of 368. Box-and-whisker plots indicate the range of scores from patients in each category (vertical lines) and the range of the first and third quartile in each category (boxes). The horizontal line inside the box indicates the median.</p

SBP1 induction results in an increase in phospho-p53 and a decrease in total p53 in HCT116 cells.

<p>Total cell extracts from doxycycline treated or non-treated HCT116-TetSBP1 cells were analyzed using immunobloting for changes in phosphorylated Ser15 on p53 (A) and total p53 (B) levels in response to induction of SBP1 using anti-human SBP1, phospho p53-Ser15, p53, and β-Actin antibodies. β-Actin was used as an endogenous control.</p