Table 1.
Clinicopathologic Characteristics of Validation Cohort.
Fig 1.
GLB1 increases expression in HGPIN and PCa.
(A) Gene expression was measured using the AQUA platform as described [20] in an initial multitissue prostate array. Epithelial GLB1 expression is significantly increased in HGPIN, primary prostate cancer and BPH compared to benign in both nuclear and cytoplasmic compartments (All p<0.001). For all tissue types, expression was 2–3 times greater in the nucleus than the cytoplasm (p<0.0001). (B) Tissue microarrays immunofluorescently stained Glb1 (red) primarily in the epithelium.
Fig 2.
GLB1 is increased in replicative and therapy-induced senescent (TIS) cell culture models.
(A) Human prostate epithelial cells were cultured to senescence by repeated cell passaging demonstrate an enlarged, more complex senescent cell morphology and increased β–gal staining in passage 5 (P5) than in proliferating (P1) cultures. (B) Western blot comparing proliferating HPECs (P1) versus later passages (P5) for separate cultures (S1, S2, S3) are shown. GLB1 and p16 expression is increased in senescence. Cultures contained >50% senescent cells by SA-βgal staining. Images were normalized to α-tubulin. (C) GLB1 mmunofluorescence increases in cancer cells displaying TIS. Du145 and LNCaP cells were treated with 250nM AZQ or DMSO (control) for 3 days, kept in drug-free media for an additional two days, then stained with mouse anti-human GLB1 antibody and detected with anti-mouse-Alexa 647. (D) Western blot analysis of GLB1 expression at 5 or 7 days after AZQ treatment performed in DU145 and LNCaP PCa lines. Cells were compared against a DMSO-treated control group (Con) and normalized to α-tubulin expression levels. (E) Cell granularity and complexity analysis of senescence by flow cytometry. Du145 cells were treated with AZQ or Doxorubicin for 3 days, kept in drug-free media for two days then collected and applied to MACSQuant flow cytometer immediately. Side scatter (SSC), which increases in senescent cells, increased in senescent AZQ and Doxorubicin treated cells compared to control.
Fig 3.
GLB1 increases expression in HGPIN and a subset of PCa and correlates with decreased proliferation and increased expression of HP1γ.
Gene expression was measured using the Vectra platform in a larger tissue array. In all tissue types, nuclei stained more strongly for GLB1 than the cytoplasm (p<0.0001). Data are mean +/- 95% confidence intervals. * p<0.001. (A) Epithelial GLB1 expression is significantly increased in HGPIN and cancer compared to benign prostate in both nuclear and cytoplasmic compartments (All p<0.0001). (B) Stromal GLB1 expression levels in HGPIN and cancer exhibited similar intensities of staining (p<0.001 all). (C) The mean GLB1 expression was calculated for all samples and the number of cores that overexpressed GLB1 was determined for each pathologic entity. Primary cancer samples demonstrated increased expression in a subset of samples (nuclear expression shown). (D) The microarrays were immunostained for the proliferation protein Ki67, the heterochromatin protein HP1γ increased in senescence, and p27, a marker decreased in terminal senescence. Separating HGPIN cores into high and low Ki67 and GLB1 staining, the population of high GLB1-low Ki67 (right), representing the senescent population, expressed low p27 and high HP1γ as expected for senescent cells. This was significantly different from the expression of p27 and HP1γ in the low-GLB1-high Ki67 population (left), confirming the association of GLB1 with other senescent markers in vivo. Other subpopulations (high GLB1-high Ki67 and low GLB1-low Ki67) did not demonstrate significance and were omitted for clarity.
Table 2.
Association of GLB1 with Clinicopathologic Characteristics in Primary PCa Samples.
Fig 4.
Increased GLB1 identifies patients at decreased risk of PCa recurrence.
GLB1 expression on a per-core basis was measured using the Vectra platform in all tissues. Patients were stratified into high or low GLB1 levels based on median core expression. Kaplan-Meier survival curves are shown. (A) Increased GLB1 expression is associated with longer PSA-free recurrence in PCa patients. Stratified by low (n = 12) and high GLB1(n = 32) expression, cumulative PSA-free survival significantly increases in patients who had high GLB1 levels in a log rank analysis (p = 0.025). (B) High (n = 20) and low (n = 7) GLB1 levels predict PSA-free survival times (629 vs. 2314 days, respectively; p = 0.013) in intermediate Gleason Score (GS 6–7) patients. (C). Per cell GLB1 expression is more homogenous in lower grade PCa tissues which express increased GLB1. GLB1 expression on a per-cell basis was then measured using the Vectra platform in all tissues. HGPIN had the least heterogeneity (p<0.001). Primary cancer tissue overall has significantly less heterogeneity than benign (p<0.001). Primary cancer cells with associated metastatic disease (Met+) had lower levels compared to localized tumors without the presence of metastatic (Met-) disease (p = 0.001) (D). Mean nuclear GLB1 expression was inversely correlated with the coefficient of variation (representing heterogeneity) of the cores (Pearson's r = -0.573; p<0.01; N = 265). Tissues that display the highest levels of GLB1 staining have the most homogenous staining.