**4. Discussion**

Tumours are able to diversify their microenvironment and consequently the altered, more reactive tumour microenvironment can modulate the response of tumours to therapy treatment [24–28]. Herein, activated stromal cells and in particular activated fibroblasts/CAF can mediate therapy resistance of malignant epithelial cells in a CAV1-dependent fashion [4,5,29,30].

CAV1-dependent stromal-epithelial crosstalk in tumours with the potential to induce resistance includes processes such as autophagy or the 'reverse Warburg effect' [31,32]. For example, a more reactive stromal phenotype following a decrease of CAV1 expression by lysosomal degradation in fibroblasts was observed when cancer cells induced oxidative stress in the tumour-microenvironment [33]. In turn, downregulation of CAV1 in fibroblasts leads to increased oxidative metabolism in cancer cells, fostering cell resistance [29]. Importantly, extrinsic factors from the microenvironment and in particular from activated fibroblasts/CAF, may drive resistance in a non-tumour cell autonomous mechanism [34,35]. In line with these findings, we have recently shown that CAV1-deficient fibroblasts mediate radiation resistance of human prostate carcinoma cells *in vitro* and *in vivo* and that the decrease in cell death after radiation treatment is mediated though a paracrine mechanism of action [11]. However, the exact resistance-promoting effectors, as well as the role of CAV1-dependent fibroblast-derived factors, remained elusive. We therefore hypothesized that fibroblast-derived inhibitors of apoptosis proteins could mediate cell death resistance upon radiation. Here we show that TRIAP1 is highly expressed in stromal fibroblasts in a CAV1-dependent manner. *In vitro*, an ectopic expression of TRIAP1 leads to a cell specific increased radiation resistance in p53-deficient PC3 and p53-wildtype LNCaP prostate cancer cells, whereas p53-mutant DU145 cells do not gain any radiation resistance. Conformingly and mimicking the human situation more precisely, induced over-expression of TRIAP1 in human prostate fibroblasts leads to induced radiation resistance. Further on, TRIAP1-expressing stromal fibroblasts mediate radiation resistance *in vivo* when respective cells are co-implanted with CAV1-deficient PC3 tumour cells.

The underlying mechanism by which fibroblast-derived TRIAP1 is secreted and subsequently taken up by adjacent cancer cells and/or shuttled between the stromal and the tumour cells needs to be investigated further. TRIAP1 secretion in fibroblasts with a reduced CAV1-content is paralleled by the presence of lysosomal exocytosis related proteins and enzymes, such as ASM, ASA and LAMP proteins. This indicates that fibroblasts with a reduced CAV1 content bear a higher lysosomal exocytosis activity compared to fibroblasts containing normally high amounts of CAV1. It is known that the process and regulation of lysosomal exocytosis is largely changed upon tumour progression and in transformed cells [36]. Released lysosomal hydrolases, such as cathepsins D and B, play a role in tumour growth invasion and angiogenesis [37]. LAMP2 contributes to resistance, as the so called lysosomal cell death induced by anti-cancer drugs is decreased when LAMP2 is overexpressed in fibroblasts [38]. In addition, ASM is down-regulated in several carcinomas, for example, head and neck cancer and gastrointestinal carcinoma cancer cells, leading to a destabilized lysosomal environment in combination with an anti-apoptotic adaptation by decreased ceramide production [36]. Lysosomal exocytosis in cancer cells has been suggested to facilitate the entrapment and clearance of chemotherapeutics and provide an additional line of resistance [39].

As intrinsic drug resistance might be caused, at least in part, by factors secreted by the tumour microenvironment, it is thus imperative to dissect the tumour-microenvironment interactions which may reveal important mechanisms underlying drug resistance [35,39].

Interestingly, immunohistological analysis of TRIAP1 in advanced human prostate cancer reveals increased TRIAP1 immunoreactivity in the malignant epithelial cells of the more radioresistant higher Gleason grade adenocarcinomas. This highlights fibroblast-derived TRIAP1 as a potential candidate for future CAV1-mediated radiation response modulation. TRIAP1 is also involved in prostate cancer bone metastasis [40] and sensitivity to doxorubicin in breast cancer cells [15]. In ovarian cancer cells, increased TRIAP1 levels correlate with increased proliferation, a decrease in apoptosis and overall tumour progression [41]. TRIAP1 is also found to be upregulated in multiple myeloma [42], and, in patients with nasopharyngeal carcinoma, TRIAP1 overexpression correlates with a poor survival rate [43]. Experimental knockdown of TRIAP1, by expression of micro RNA miR-320b, is able to induce apoptosis by mitochondrial deregulating mechanisms, such as cytochrome C release and membrane potential alterations [15,43].

In summary, we have specified the role of CAV1 alterations potentially induced by CAV1-deficient and more reactive, stroma in radio sensitivity of prostate carcinoma at molecular level. We have identified apoptosis inhibitor TRIAP1 as a stromal-derived factor with the potential to induce cancer cell resistance. We sugges<sup>t</sup> that blocking TRIAP1 activity and avoiding drug resistance may offer a promising drug development strategy to inhibit resistance-promoting CAV1-dependent signals.

**Author Contributions:** J.K., A.P., A.W. and D.K. performed experiments; J.K., D.K. analysed results and made the figures; C.H., P.M., E.Y. and H.R. provided materials; H.R. performed the Gleasing scoring; D.K. and V.J. designed research, J.K. and D.K. wrote the paper and E.Y. performed language corrections. All authors reviewed and approved the manuscript. This work was supported by grants of the DFG (GRK1739/1; GRK1739/2) and the BMBF (02NUK024-D).

**Acknowledgments:** We thank Mohammed Benchellal and Eva Gau for their excellent technical assistance. We acknowledge support by the Open Access Publication Fund of the University of Duisburg-Essen.

**Conflicts of Interest:** The authors state that there are no personal or institutional conflict of interest.
