**3. Results**

### *3.1. Radioresistant (CAV1-Silenced) Fibroblasts Express and Secrete Anti-Apoptotic TRIAP1*

We previously reported that CAV1-deficient fibroblasts foster radiation resistance of malignant prostate epithelial cells resulting in decreased apoptosis rates *in vitro* and *in vivo*, most likely via a paracrine mechanism of action [11]. Because we hypothesized that fibroblasts could allocate CAV1-dependent apoptosis inhibiting proteins to the tumour cells, we investigated the presence and expression levels of well-known resistance-associated anti-apoptotic proteins in stromal HS5 fibroblasts being either proficient [HS5(+)] for CAV1 or CAV1-deficient [HS5(-)] achieved by a shRNA-mediated knock-down (Figure 1). Of note, CAV1-silenced HS5 fibroblasts expressed significantly higher levels of TRIAP1 at both protein (Figure 1A) and mRNA level (Figure 1B). In addition, an increased CAV1-dependent TRIAP1 secretion was confirmed in cell culture supernatants of CAV1-silenced HS5 fibroblasts, which was accompanied by increased levels of lysosomal enzymes (acid sphingomyelinase, ASM and arylsulfatase A, ASA), which might be indicative for lysosomal exocytosis (Figure 1C).

**Figure 1.** Radiation-resistant Caveolin-1 (CAV1)-silenced fibroblasts differentially express and secrete the apoptosis inhibiting protein TP53-regulated inhibitor of apoptosis 1 (TRIAP1).

Thus, increased expression and secretion of TRIAP1 by CAV1-silenced fibroblasts suggests that secreted TRIAP1 and then internalized by neighbouring prostate cancer cells, might account for the induced radiation resistance of these cells.

(A) Protein expression levels of apoptosis inhibiting proteins survivin, XIAP (X-linked inhibitor of apoptosis protein) and TRIAP1 were determined in CAV1-proficient [Cav1(+)] and CAV1-silenced [Cav1(-)] HS5 fibroblasts. Indicated proteins were analysed in whole protein lysates 96 h after radiation with 10 Gy by western blot analysis. Representative blots are shown. For TRIAP1 quantification, blots were analysed by densitometry and the respective signal was normalized to that from β-actin (n = 3–4 for each group). *p*-values were indicated: \* *p* ≤ 0.05, \*\* *p* < 0.01 by one-way ANOVA followed by post-hoc Tukey's test.

(B) qRT-PCR quantifications of TRIAP1 mRNA levels were performed 96 h post irradiation and shown as relative expression to β-actin mRNA. Data shown represent mean values ± SEM from 4 independent samples per group, each measured in duplicate. \* *p* ≤ 0.05, \*\* *p* ≤ 0.01, by one-way ANOVA followed by post-hoc Tukey's test.

(C) TRIAP1 and lysosomal enzymes (ASM, acid sphingomyelinase and ASA, arylsulfatase A) secretion were further determined in cell culture supernatants derived from CAV1-silenced HS5(-) or control transfected CAV1-expressing HS5(+) fibroblasts with or without radiation treatment (10 Gy) using western blot analysis. Equal protein amounts (100 μg) were loaded. Ponceau S staining of transferred proteins was included as loading control.

### *3.2. Ectopic TRIAP1 Expression in Prostate Carcinoma Cells Induces Radiation Resistance*

We previously have shown that cell culture supernatants of CAV1-silenced HS5 fibroblasts were able to induce radiation resistance of PC3 and LNCaP cells by decreased apoptosis [11]. We then investigated if the induced resistance of prostate cancer cells, after treatment with supernatants derived from CAV1-proficient or -deficient fibroblasts, led to higher TRIAP1 levels (not shown). However, no increased TRIAP1 levels were detectable in PC3, DU145 or LNCaP prostate carcinoma cells upon supernatants treatment most likely because the amount of tumour cell internalized TRIAP1 which was secreted from fibroblasts did not pass the threshold level of detection by western blot analysis. To provide the proof of principle that TRIAP1 mediates radiation resistance, the prostate cancer cells PC3 (p53 null), DU145 (p53 mutant) and LNCaP (p53 wild type) were transiently transfected with an expression vector encoding for human GFP-tagged TRIAP1 (Figure 2A). Empty vector transfected cells served as a control. Ectopic TRIAP1 expression resulted in decreased subG1 levels in PC3 and LNCaP cells 48 h after radiation with 10 Gy and thus increased resistance to radiation treatment. However, DU145 cells were not affected. Increased TRIAP1-levels were confirmed by western blot analysis (Figure 2B). Cell cycle analysis further revealed that ectopic TRIAP1 expression resulted in a slightly diminished G0/G1 subpopulation in PC3 cells upon radiation, while the proportion of cells in the G2/M phase increased (Figure 2C). The cell cycle of DU145 prostate carcinoma cells after TRIAP1 transfection was not affected upon radiation. Similar to PC3 cells, more TRIAP1-transfected LNCaP cells were in the G2/M phase after radiation as compared to control transfected cells. The proportions of respective cells in the S and <4n phase were rather low and not affected (not shown).

These results indicate that ectopic TRIAP1 expression mediates radiation resistance in a cell-type dependent manner and sugges<sup>t</sup> that resistant prostate cancer cells will have an increased proliferation potential.

(A) Prostate cancer cells were transiently transfected with an expression vector encoding for human TRIAP1-GFP. Empty vector served as control. 24 h after transfection cells were irradiated with 0 or 10 Gy. The degree of apoptosis was quantified measuring the SubG1 fraction after radiation by flow cytometry analysis after additional 48 h of culture. Data shown represent mean values ± SEM from 4–5 independent samples per group measured in duplicates each. \* *p* ≤ 0.05, by two-tailed students *t*-test.

(B) Efficiency of TRIAP1-GFP expressions as analysed by Western blots. Representative blots from 3-4 independent experiments are shown. β-actin is used as a loading control. As additional control (Ctrl) mock transfected cells, which underwent the transfection procedure without an expression vector were shown.

**Figure 2.** Ectopic TRIAP1 expression in prostate carcinoma cells results in radiation resistance.

(C) Cell cycle analysis of TRIAP1-GFP transfected prostate cancer cell lines was performed using Nicoletti/PI staining and flow cytometry. Empty vector transfected cells served as control. Data represent mean values ± SEM from 3–5 independent samples per group measured in duplicates each. \*\* *p* ≤ 0.01, by two-way ANOVA followed by post-hoc Tukey's test.

### *3.3. Generation of Stromal Prostate Fibroblasts with Stable TRIAP1 Expression*

Prior to investigating whether TRIAP1 derived from a reactive tumour stroma might account for the radiation resistance observed in PC3 xenografts *in vivo* [11], we assessed the suitability of another fibroblast cell type, prostate fibroblasts (WPMY-1) derived from healthy donors, to more closely mimic the human situation in future *in vivo* experiments (Figure 3).

Compared to normal HS5 fibroblasts, WPMY-1 prostate fibroblasts expressed less endogenous CAV1-expression levels (Figure 3A). Quantitative Real Time RT-PCR analysis of TRIAP1 expression levels as well as of reactive fibroblasts markers (ACTA2 and TAGLN) and tumour-promoting EMT factor transforming growth factor β (TGFB1) in WPMY-1 fibroblasts (+/− XRT) confirmed the more reactive phenotype of WPMY-1 with a less pronounced CAV1-content and furthermore of irradiated WPMY-1 fibroblasts (Figure 3B). In line with previous findings [11], colony formation assays indicated that WPMY-1 fibroblasts with a reduced CAV1 content were more resistant to radiation (Figure 3C). To further investigate a potential TRIAP1-mediated radiation resistance of prostate carcinoma cells caused by the stromal compartment, we generated TRIAP1-overexpressing WPMY-1 fibroblasts via transfection of WPMY-1 with an expression vector encoding for human TRIAP1 tagged with GFP (Figure 3D). Stably transfected and TRIAP1-GFP-sorted cells (via flow cytometry) were successfully generated. Increased TRIAP1 expression was confirmed by western blot analysis. It is worth noting that TRIAP1-overexpression did not alter CAV1 expression levels (Figure 3D). Ectopic TRIAP1 expression resulted in a significant reduced subG1 population upon radiation, which confirmed the

resistant phenotype of TRIAP1-GFP expressing prostate fibroblasts (Figure 3E). TRIAP1 secretion from TRIAP1-GFP-expressing cells was confirmed by western blot analysis from cell culture supernatants and revealed an increased secretion upon radiation (Figure 3F).

**Figure 3.** Characterization of the human prostate fibroblast cell line WPMY-1.

(A) CAV1 expression levels analysed by western blot in normal HS5 and prostate WPMY-1 fibroblasts, with or without radiation treatment with 10 Gy (96 h post irradiation). β-actin was included as loading control. Representative blots of at least three different experiments are shown.

(B) qRT-PCR quantifications of TRIAP1 mRNA levels, as well as reactive fibroblast markers, were performed 96 h post irradiation and shown as relative expression to β-actin mRNA. Data shown represent mean values ± SEM from 4-6 independent samples per group measured in duplicate each. \* *p* ≤ 0.05, \*\* *p* ≤ 0.01, by two-way ANOVA followed by post-hoc Tukey's test.

(C) Colony formation assay of HS5 and WPMY-1 cells. Following irradiation (0–8 Gy) cells were further incubated for 10 days. Data show the surviving fractions from three independent experiments measured in triplicates each (means ± SD). \*\*\* *p* ≤ 0.005, \*\*\*\* *p* ≤ 0.001 by two-tailed students *t*-test.

(D) The degree of apoptosis was quantified measuring the SubG1 fraction 48 h after radiation by flow cytometry. Data shown indicate mean values ± SEM from 3 independent samples per group measured in duplicates each. \* *p* ≤ 0.05, by two-way ANOVA followed by post-hoc Tukey's test.

(E) WPMY-1 prostate fibroblasts were transfected with a TRIAP1-GFP encoding plasmid or empty vector in the control, selected with G418 and sorted via flow cytometry to select GFP-expressing cells. Expression levels of TRIAP1-GFP and CAV1 were confirmed by western blot analyses, with or without 10 Gy irradiations. Band signal intensity was quantified by densitometry and normalized to that from β-actin. Data represent mean ± SEM from three independent experiments. *p*-values were indicated: \*\* *p* < 0.01; \*\*\* *p* ≤ 0.005 by two-way ANOVA followed by post-hoc Tukey's test.

(F) TRIAP1-GFP secretion in cell culture supernatants derived from TRIAP1-GFP or control, transfected WPMY-1 fibroblasts with or without radiation treatment (10 Gy) determined by western blot analysis. CAV1, ASM and ASA secretion levels were also investigated. Equal protein amounts (100 μg) were loaded. Ponceau S staining of transferred proteins was included as loading control.

### *3.4. TRIAP1-Expressing Stromal Fibroblasts Mediate Radiation Resistance*

Next, we asked whether fibroblastic tumour stroma-derived TRIAP1 accounts for an increased radiation resistance in PC3 xenograft tumours [11]. To mimic the human situation we performed subcutaneous transplantations onto the hind limb of NMRI nude mice by injecting CAV1-silenced PC3(-) tumour cells in combination with control-transfected or TRIAP1-GFP-expressing WPMY-1 prostate fibroblasts (Figure 4). Prostate xenografts were implanted onto the hind limb of NMRI nude mice and were irradiated locally with a single dose of 10 Gy when the tumour reached a size of about 100 mm3 (around day 3). Tumour growth was determined by measuring the tumour volume 3 times a week (Figure 4A). Either co-implantation with WPMY-1 cells, control or TRIAP1-transfected, did not change tumour growth. The tumour growth delay after radiation was significantly decreased in PC3(-)-derived tumours co-implanted with TRIAP1-expressing WPMY-1. These tumours showed a significantly increased growth after radiation treatment when compared to PC3(-)-derived tumours co-implanted with control-transfected WPMY-1 as demonstrated by the reduced time to reach a four-fold tumour volume (Figure 2B). Immunohistochemistry using the proliferation marker PCNA (proliferating cell nuclear antigen) antibody further confirmed an increase in the proliferation rate of PC3(-) xenografts when co-implanted with TRIAP1-GFP expressing fibroblasts and a significantly decreased sensitivity to radiation treatment (Figure 4C). Thus, in line with the *in vitro* results, TRIAP1 derived from stromal fibroblasts is able to induce radiation resistance of prostate tumours.

**Figure 4.** TRIAP1 expression in fibroblasts fosters radiation resistance in tumours derived from PC3 CAV1(-) cells.

(A) PC3 CAV1(-) cells were subcutaneously injected with either TRIAP1-expressing WPMY-1 or control-transfected fibroblasts (0.5 × 10<sup>6</sup> cells in total, ratio1/1) into the hind limb of NMRI nude mice. One set of animals from each group received a single tumour radiation dose of 10 Gy once its growth was easily detected (around day 3). Tumour volume was determined at indicated time points using a sliding calliper. Data are presented as mean +/ − SEM from 3 independent experiments (26 mice in total: Ctrl 0 Gy n = 6; Ctrl 10 Gy n = 7; TRIAP1 0 Gy n = 6; TRIAP1 10 Gy n = 7).

(B) Tumour growth (*left panel*) and respective computed median growth delay (*right panel*) were determined as time (days) until a four-fold tumour volume was reached. \*\*\* *p* < 0.005; \*\*\*\* *p* < 0.001 by one-way ANOVA followed by post-hoc Tukey's test.

(C) Immunohistochemical analysis of TRIAP1, PCNA and CAV1 in isolated PC3 xenograft tumours. Sections were counterstained using haematoxylin. Representative images are shown. Magnification 200×, scale bar 50 μm (left panel), magnification 400×, scale bar 20 μm (right panel).

### *3.5. Human Advanced Prostate Cancer Specimens Were Characterized by an Increased TRIAP1-Immunoreactivity Indicating Radiation Resistance*

As loss of stromal CAV1 is paralleled by a radiation-resistance promoting reactive tumour stroma in human prostate tissue specimens [11,21], we decided to investigate TRIAP1 expression levels, as well as the respective stromal-epithelial TRIAP1 distribution, in human prostate tissue specimens by immunohistochemistry. TRIAP1 expression in prostate epithelial cells increased with higher Gleason scores, that is, lower tumour differentiation (Figure 5). Furthermore, stromal cells of tumour samples tended to be more intensively stained in cases with higher Gleason grade (Figure 5). These results indicate that increased, potentially fibroblast-derived, TRIAP1 has implications for prostate carcinoma progression and therapy resistance.

**Figure 5.** Immunohistochemical analysis of TRIAP1 expression levels in human prostate cancer tissues.

Paraffin-sections of human prostate cancers were stained for TRIAP1. Gleason grading scores were divided into low (Gleason Score ≥ 6, Grade group 1), intermediate (Gleason Score 7 (a/b), Grade groups 2 & 3) and high scores (Gleason Score ≥ 8, Grade groups 4 & 5). Asterisks mark stromal compartments and bold arrows point to epithelial structures. Sections were counterstained

using haematoxylin. Representative images are shown. Magnification 200x. Right panel: higher magnification images: 400x.
