Discussion

The discovery of novel CRC biomarkers to assist in early diagnosis, prognostic stratification and prediction of response to treatment is an urgent medical need. Immunohistochemistry has been routinely used to assess the expression of proteins with prognostic or predictive value in other types of cancer such as breast (Zaha, 2014) and lung carcinomas (Pekar-Zlotin et al., 2015), soft tissue sarcomas (Hornick, 2014) and lymphomas (Higgins et al., 2008). Besides, it is also an essential technique in the attempt to elucidate the origin of metastatic cancer from an occult primary site (Conner and Hornick, 2015, Oien and Dennis, 2012). In CRC, immunohistochemistry has mainly been used to identify the colorectal origin of metastatic lesions based of the presence CDX (an intestine specific transcription factor) and cytokeratin 20, and the absence of cytokeratin 7 (Coghlin and Murray, 2015). Mismatch repair deficiency (a surrogate marker for hereditary non-polyposis colorectal cancer) is also assessed using MLH1 and MSH2 immunostaining and it seems that the status of these mismatch-repair proteins in sporadic CRC has a prognostic role (Coghlin and Murray, 2015). Apart from these limited examples, no robust IHC biomarker has been validated for use in clinical practice. It highlights the importance of discovering new tools to better stratify cases and improve outcomes from treatment. Additionally, the definition of low-risk and high-risk adenomas is currently based on microscopic architecture, i.e. grade of dysplasia (Risio, 2010) and endoscopic features such as size, shape and number (Kurome et al., 2008). The discovery of an immunohistochemical profile to help accurately identify lesions with a higher risk of progression could potentially improve the management of individuals with colorectal polyps.

Mutations resulting in inactivation of APC or over-activation of β- catenin (both leading to the activation of Wnt pathway) are the earliest and

164 most common genetic defects in human CRC (Cancer Genome Atlas Network, 2012). Our group has hypothesised that the study of animal models of CRC based on Apc gene inactivation could lead to the discovery of novel useful cancer biomarkers. For this purpose, a model of acute Apc inactivation (AhCre+ _Apcfl/fl _{mouse) and a model of chronic Apc deletion (Apc}Min/+ _mouse) have previously been studied by our group (Hammoudi et al., 2013, Ibrahim, 2014). This work has resulted in a list of potential candidate biomarkers which have now been subjected to a validation study using human samples.

In this study, we tested different IHC scoring systems to assess the expression of our candidate biomarkers in different pathological stages of the adenoma-carcinoma sequence and have used samples from two geographically distinct populations. Our first finding is that, independent of the scoring method used or the population assessed, the results were highly consistent. This shows that either the manual or the electronic scoring systems used are suitable for the evaluation of our candidate proteins. Additionally, it suggests that the expression pattern of the potential biomarkers is likely to be similar in different ethnic groups, as was demonstrated in this analysis using two different populations. This is important because specific genetic alterations or the expression of biomarkers may vary across different populations. For example, it has been shown that EGFR mutations in sporadic lung cancer are more common in Asian patients (Shigematsu et al., 2005) and BRCA germline mutations (resulting in increased risk of several types of cancer, especially breast carcinomas) are more prevalent in Ashkenazi Jews (Struewing et al., 1997).

As our candidate biomarker list was originally derived from animal models of Apc inactivation, our initial task was to assess Wnt signalling pathway activation in our samples. This was clearly demonstrated (in the form of β-catenin cytoplasmic and nuclear localisation) in neoplastic lesions from low-grade adenomas to invasive cancers and our findings were concordant with the literature (Wong et al., 2004, Wong et al., 2003, Chen et al., 2013). Next, we assessed the remaining candidates using both a manual modified-H score, and electronic nuclear and cytoplasmic scores (ImmunoRatio and IHC Profiler plugins, respectively). Results obtained for

165 NAP1L1 and RPL6 revealed an opposite pattern of immuno-expression when compared to β-catenin. Both proteins exhibited a clear decrease in nuclear expression in neoplastic tissues compared to the adjacent mucosa. Interestingly, the expression of NAP1L1 and RPL6 in low-grade adenomas was similar to that observed in the adjacent normal mucosa (UK-cohort) and normal control samples (Brazilian cohort), as opposed to β-catenin which exhibited an altered staining pattern even in low-grade adenomas. These findings suggest that both NAP1L1 and RPL6 might be useful immunohistochemical biomarkers for colorectal cancer and for high-grade (high-risk) adenomas. No published study assessing the immuno-expression of these two proteins in CRC tissues or adenomas has been found. Therefore, our data provide the first description of the IHC pattern of expression for both biomarkers in this context and suggest a possible role for these proteins in determining the progression from low-grade to high-grade adenomas and the subsequent development of CRC.

Another candidate that showed a clear differential expression pattern in our study was Prohibitin (PHB). As was the case for NAP1L1 and RPL6, PHB showed decreased nuclear staining in colorectal cancer tissues compared to the adjacent mucosa whereas an increase in cytoplasmic staining was observed. This interesting finding suggests that the protein might be displaced from the nucleus towards the cytoplasm during malignant transformation. Again, these alterations were specifically seen in cancer tissues and not in low-grade adenomas. Increased cytoplasmic expression of PHB in CRC has also been demonstrated in another study (Chen et al., 2010a). Taken together, these results support the potential use of PHB as a CRC biomarker.

HMGB1 is a multifunctional protein involved in many disease conditions (Kang et al., 2014). Its role in carcinogenesis has been a subject of scientific debate (Kang et al., 2013). In CRC tissues, HMGB1 immuno- expression has been extensively studied and mixed results have been published (Süren et al., 2014, Zhang et al., 2015a, Ueda et al., 2014, Lee et al., 2012, Peng et al., 2010). Our analysis performed using electronic scoring tools in cancer and adjacent tissues from the Brazilian cohort did not

166 demonstrate any difference in HMGB1 expression between the groups. A uniformly positive nuclear staining was observed in both tumour and adjacent tissues, whilst a negative cytoplasmic staining was seen in both conditions. These results are concordant with the findings from Peng et al. who demonstrated that all of the adjacent tissues in their CRC cohort exhibited positive nuclear and negative cytoplasmic HMGB1 staining. In the cancer counterpart, the same pattern was observed in 81.5% of cases (Peng et al., 2010).

SFRS2 and CDC5L were suggested as potential biomarkers of CRC in our prior study (Ibrahim, 2014). Neither of these proteins has been previously evaluated in CRC tissues. Our assessment of SFRS2 and CDC5L immuno- expression showed a very small increase in nuclear staining for both proteins. Although statistically significant, the small magnitude of these differences means that the findings are very unlikely to be clinically relevant.

The cause of this “switching” pattern of expression observed with NAP1L1, RPL6 and PHB (which seem to exit the nucleus during malignant transformation) is not clear. Gene expression may be altered secondary to Wnt pathway activation as occurs with MYC (He et al., 1998, Myant and Sansom, 2011) or protein localisation may be affected by post-translational modifications, as in the case of β-catenin following APC inactivation (Clevers and Nusse, 2012). Alternative splicing could also be involved in this setting, causing the alterations that we observed.

Altogether, our findings suggest that potential cancer biomarkers derived from animal models of Apc inactivation may yield valid candidates for human CRC. NAP1L1, RPL6 and Prohibitin immuno-expressions were consistently altered in malignant and high-grade premalignant colorectal neoplasms in a clear, reproducible and statistically significant manner. Whether the immuno-expression patterns of these proteins can be used as diagnostic tools or for risk stratification requires further investigation. The possibility of using these markers to identify adenomatous polyps which have a higher risk of progression to invasive cancer is particularly attractive. However, the assessment of the suitability of these proteins for this purpose

167 requires a larger longitudinal study and is beyond the remit of the present work. In the next chapters, I will further describe the assessment of the expression of these biomarkers using different methodologies and different matrices such as blood and frozen tissues. In addition, I will explore the prognostic importance of these proteins in CRC. As HMGB1, SFRS2 and CDC5L were not significantly altered in neoplastic colorectal tissues compared to the adjacent normal mucosa they have not been further assessed in the next steps of this research.

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Chapter Four:

Study of the RNA expression of

the biomarkers in human

samples and the effects of gene

silencing on a CRC cell line

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4. CHAPTER 4 – STUDY OF THE RNA EXPRESSION OF THE