There are a number of proteomic studies that have attempted to identify biomarkers associated with radiotherapy resistance. For each study the discovery data has been reviewed and human proteins identified using MS/MS have been assimilated by gene name in Appendix A. PMF data appears in Appendix A only if confirmatory techniques (e.g. Western blotting) were used to demonstrate the correct identification and differential expression of the protein. Those proteins which have undergone clinical validation by IHC are highlighted in Appendix A and the details given in section 3.7.2 (Scaife et al., 2011).
3.7.1 Clinical tissue studies
Owing to the technical challenges associated with clinical tissue analysis, the majority of proteomic studies of radiotherapy resistance have been carried out on cell line models. However, a single study to identify biomarkers of radiotherapy resistance using clinical tissue has been described (Allal et al., 2004). Tissue biopsy samples were collected from 17 rectal cancer patients with T2-T3/N0-N1 tumours prior to fractionated RT treatment. Following a total dose of 50 Gy, the tumour response was assessed histopathologically. RR and RS tumour samples where then compared by proteomic analysis using 2DE (pH range 4.5-5.5 and 5.5-6.7) and PMF. The putative identity of several DEP’s was reported, including annexin V (ANXA5), Kv channel interacting protein 3 (calsenilin; KCNIP3), tropomodulin 3 (TMOD3) and RAD51-like 3 (RAD51L3). No further work was carried out to confirm the identity and differential expression of these proteins.
3.7.2 Cell line studies
The majority of studies that have employed comparative proteomic methodologies in order to identify putative biomarkers associated with radiotherapy resistance have utilised novel radioresistant (RR) cancer cell lines as clinically relevant in vitro models. Established cancer cell lines can be subjected to fractionated doses of ionising radiation mimicking the relevant clinical schedule and total dose, in order to generate novel cell sub-lines that demonstrate a significant increase in radiotherapy resistance. It is hypothesised that the fractionated sub-lethal radiation dose will drive the selection of cell clones that carry RR properties and the abnormal constitutive (in)activation of key proteins associated with the
58 RR phenotype. The current collection of such published studies is outlined in chronological order below and the putative biomarkers identified have been assimilated in Appendix A.
A derivative RR sub-line of the H69 small cell lung cancer cell line was produced following a fractionated total radiation dose of 37.5 Gy (Henness et al., 2004). Differences in protein expression in this H69/R38 RR sub-line, compared with untreated parental cells, were then examined using 2DE (pH range 3-10) and MS/MS. The identities of nine human DEP’s were reported (Appendix A).
A derivative RR sub-line of the MCF-7 breast cancer cell line was produced following a fractionated total radiation dose of 60 Gy (Wang et al., 2005). Differences in protein expression in this MCF-7+FIR30 RR sub-line, compared with RS MCF-7 cells, were analysed using 2DE (pH range 3-10 and 4-7) and MS/MS (Appendix A). The identity of peroxiredoxin II was reported as a differentially expressed protein and further analysis was concentrated on this protein. The up-regulation of peroxiredoxinII in the MCF+FIR30 RR sub-line was confirmed by Western blotting and gene silencing using siRNA restored partial radiosensitivity.
Derivative RR sub-lines of the LNCaP, PC3 and Du145 prostate cancer cell lines were produced following a total radiation dose of 10 Gy (Skvortsova et al., 2008). Differences in protein expression in the LNCaP-IRR, PC3-IRR and Du145-IRR RR sub- lines, compared with the relevant parental cells, were assessed using 2D-DIGE (pH range 3-10) coupled with MALDI-TOF/TOF-MS. The identity of over 20 human DEP’s, which were observed in membrane and cytosol sub-fractions from all three RR sub-lines, was reported (Appendix A). The differential expression of APEX1, HSPA8, NME1, RAB11A and SERBP1 was validated by Western blotting. Furthermore, gene silencing of APEX1 by siRNA demonstrably enhanced radiosensitivity in all three of the RR cell sub-lines.
In our own group, derivative RR sub-lines of the MCF-7, MDA-MB-231 and T47D breast cancer cell lines were produced following a fractionated total radiation dose of 40 Gy (Smith et al., 2009). Differences in protein expression in the MCF-7RR, MDA-MB-231RR and T47DRR sub-lines, compared with relevant parental cells, were analysed using both iTRAQ and 2DE (pH range 4-7 and 7-10) combined with MALDI-TOF/TOF-MS. A small number of 2DE spots that were identified by PMF (MALDI-TOF-MS) and subsequently validated by Western blotting or RTqPCR were also described. In total the identity of over 50 human DEP’s, which were observed in at least one of the three RR sub-lines, were
59 reported (Appendix A). The differential expression of 11 putative biomarkers was confirmed by Western blotting or RTqPCR, 2 of which were clinically validated. A number of proteins were associated with the 26S proteasome and a pilot immunohistochemical analysis of archival laryngeal cancers confirmed that the decreased expression of the 26S proteasome correlated with radiotherapy resistance.
A derivative RR sub-line of the CNE2 nasopharyngeal cancer (NPC) cell line was produced following fractionated radiation (Feng et al., 2010). Differences in protein expression in the CHE2-IR RR sub-line, compared with parental cells, were assessed using 2DE and MS/MS. The identities of over 20 human DEP’s were reported (Appendix A). The differential expression of HSPA5 (GRP78), SERPINB5, SFN (14-3-3 σ) and SOD2 was validated by Western blotting. In addition, the in vitro silencing of SFN (14-3-3 σ) by siRNA was associated with increased radiotherapy resistance. A pilot immunohistochemical analysis of archival NPC samples confirmed that the downregulation of SFN (14-3-3 σ) and SERPINB5 expression correlated with radiotherapy resistance, whilst the upregulation of HSPA5 (GRP78) and SOD2 expression correlated with radiotherapy resistance. This four-biomarker panel demonstrated 90% sensitivity and 88% specificity for the prediction of radiotherapy resistance in NPC samples.
Derivative RR sub-lines of the OECM1 (gingival epidermoid carcinoma) and KB (oral epidermoid carcinoma) cell lines, which are sub-types of head and neck cancer (HNC), were produced following a fractionated total radiation dose of 60 Gy (Lin et al., 2010). Differences in protein expression in the OECM1-RR and KB-RR RR sub-lines, compared with the relevant parental cells, were assessed by pre-fractionation and 1-DE prior to identification of differentially expressed protein bands by peptide mass fingerprinting. The putative identity of 64 proteins was described from the membrane, cytosol or nuclear sub-fractions and 6 underwent further confirmatory work (Appendix A). The significant differential expression of HSPD1 (HSP60), HSPA5 (GRP78), RAB40B, HSP90B1 (GRP94, GP96) and GDF15 was confirmed by RT-PCR in both RR cell lines. Further, gene silencing of HSP90B1 by siRNA demonstrably enhanced radiosensitivity in HNC cell lines and in tumour xenografts. Interestingly, the same group had previously identified the differential expression of HSP90B1 (GRP94, GP96) in RR cell lines of NPC origin using expression microarray analysis (Chang et al., 2007).
60 A derivative RR sub-line of the Hep-2 laryngeal cancer cell line was produced following a fractionated total radiation dose of 60 Gy (Kim et al., 2010). Differences in protein expression in this RR-Hep-2 RR sub-line, compared with parental cells, were analysed using 2-DE (pH 4-7) and PMF. The putative identity of 16 proteins was described and these underwent further confirmatory work. The significant differential expression of 12 DEPs was demonstrated visually by Western blotting or RT-PCR in the RR cell line (Appendix A). Further analysis of CLIC1 by RT-qPCR, confocal microscopy and chemical inhibition established a functional role for this protein in the acquisition of the RR phenotype (Kim et al., 2010).
Derivative RR sub-lines of the FaDu and SCC25 head and neck carcinoma cell lines were produced following a total radiation dose of 100 Gy (Skvortsov et al., 2011). Differences in protein expression in the FaDu-IRR and SCC25-IRR sub-lines, compared with the relevant parental cells, were assessed using 2D-DIGE (pH range 3-10) coupled with MALDI-TOF/TOF-MS. The identity of over 30 DEP’s from both IRR sub-lines were reported (Appendix A).
In summary, it is clear from Appendix A that a large number of human DEP’s have been identified in RR cell lines through use of proteomic techniques, some of which have been further confirmed using Western blotting, transcript analysis, RNA interference or immunohistochemistry. However, when comparing this list of putative biomarkers with those discussed in Chapter 2 and those hypothesised in Table 1 (Chapter 1) there is very little overlap in relation to individual biomarkers, pathways or common themes. In addition, none have yet been brought into routine clinical use, highlighting the need for increased research into the search for predictive biomarkers of radiotherapy resistance using proteomic methodologies.
61