Figure 5. OC2 promotes mCRPC tumor growth and metastasis.

(a) Cell survival 4 days after start of OC2 depletion. The mean + S.D. from three independent experiments is shown. Unpaired two-tailed Student´s t-test, C4–2: sh1^★★P= 7.58×10⁻⁵, sh2^★★P= 7.48×10⁻⁵; LNCaP: sh1^★★P=1.57×10⁻⁶, sh2^★★P=2.03×10⁻⁶, 22Rv1: sh1^★★P=5.42×10⁻⁶, sh2^★★P= 0.0002.

(b) OC2 depletion results in cell death 4 days after infection, measured by flow cytometry and double staining with Annexin V-FITC and propidium iodide (PI). These experiments were repeated independently three times with similar results.

(c) Growth in cell culture of 22Rv1 cells after OC2 depletion. The mean +/− S.D. from three independent experiments is shown. Unpaired two-tailed Student´s t-test, sh1^★★ P=0.0006, sh2^★★P=0.0009.

(d) Growth in soft agar of 22Rv1 cells after OC2 depletion. The mean + S.D. from triplicates is shown. The result shown is representative of two independent experiments.

(e) Subcutaneous 22Rv1 tumor growth after OC2 depletion (left). Appearance of tumors after 31 d (top right). Tumor volume (middle) and weight (bottom right) are shown as mean + S.D. (N=10 per mice group). Unpaired two-tailed Student´s t-test, tumor volume ^★★P=0.005, tumor weight ^★★P=0.0002.

(f) Effect of OC2 silencing on 22Rv1 metastasis following intracardiac injection. Bioluminescence images of mice bearing metastatic tumors 5 weeks after injection (top) and graphical representation of normalized bioluminescence signals of tumor development (bottom). N=9 mice per group. The boxes show the 25^th-75^th percentile range and the center line is the median. Whiskers include the smallest and largest values. Wilcoxon two-tailed rank-sum test, in week 3 ^★★P= 0.023, in week 4 ^★★P= 0.009, in week 5 ^★★P=0.0017.

(g) Association of BCR-free survival with OC2 expression. High (N=46) and low (N=45). Cox proportional hazard regression, two-sided test, P=0.002.

(h) Inverse correlation between OC2 activation and AR activation based upon mRNA expression in tumor foci of diagnostic prostate needle biopsies (N=99). Spearman’s rank correlation test, P=3.7×10⁻⁵⁷.

(i) Ratio of OC2 activation to AR activation in diagnostic prostate needle biopsies (M0-MN (N=23) and M1-Poly (N=52)). M1-Poly = patients with concurrent polymetastases (>5 bone or extrapelvic lymph node metastases and/or visceral metastases) at the time of diagnostic biopsy. M0-NM = high-grade prostate tumor biopsies from patients that remained locally confined (no metastatic progression) with average follow-up of 56 months. The boxes show the 25^th-75^th percentile range and the center line is the median. Whiskers show 1.5 times the IQR from the 25^th or 75^th percentile values. Data points beyond the whiskers are displayed using dots. Wilcoxon two-tailed rank-sum test, P=0.043.

(j) Expression of OC2 mRNA in benign and high risk primary prostate tumors from Wyatt et al.³⁷. NHT= neo-adjuvant hormone therapy.