Especificación de los casos de uso

Radiotherapy is frequently applied as a technique for the management of metastatic cSCC (see Chapter 1) and may impact the gene expression profiles of surviving cells. On a global level, the gene expression profile of UW-CSCC1-R was different to that of UW-CSCC1, as evidenced by further downregulation across most pathways. When looking at individual genes, there were in fact more differentially expressed genes (on the basis of ≥2 or ≤-2 fold- change and P < 0.05) unique to UW-CSCC1 than UW-CSCC1-R, compared to the originating tumour. Whilst this would seem contrary to how UW-CSCC1-R clusters further away from the clinical tumour than UW-CSCC1 does in a pathways heatmap, this is due to the heatmap taking into account the overall pathway scores. Assigning fold-change thresholds such as ± 2

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also ignores all genes with slightly smaller changes. This is impactful upon our interpretations as UW-CSCC1-R possessed a greater number of genes with moderate (yet not significant) differences in gene expression, totalling to a larger observable difference in the pathway score.

3.4.5.1 Cell adhesion

Downregulation of cell adhesion genes was clear in UW-CSCC1-R and corresponds with observations in cell culture (Chapter 2). The lower expression of DLC1 in UW-CSCC1-R may contribute to reduced cell adhesion as this tumor suppressor is central to the formation of focal adhesions (Kim et al., 2007; Tripathi and Lowy, 2017). E-cadherin (CDH1) and

EPCAM gene expression was significantly lower in UW-CSCC1-R. This gene is responsible

for adherens junctions between cells and is often used as an epithelial marker (Batlle et al., 2000; Mani et al., 2008). A loss of E-cadherin expression along with an increase in neural cell adhesion molecules (e.g. NCAM1) as shown in UW-CSCC1-R is generally reflective of collective migration, also referred to as incomplete EMT (Lee et al., 2006; Lehembre et al., 2008; Friedl et al., 2012). This aligns with the observations of collective cancer migration within the organotypic assay for UW-CSCC1-R (Chapter 2).

The tumour suppressor gene THY1 is downregulated in UW-CSCC1-R. This gene has been shown to mediate melanoma attachment to endothelium (Rege and Hagood, 2006), and as such, may be implicated in cell adhesion in cSCC. Additionally, the decreased expression of

THY1 could be evidence of further tumour suppressor mutation (Abeysinghe et al., 2003),

enabling UW-CSCC1-R survival and proliferation.

The largest difference between UW-CSCC1 and UW-CSCC1-R was observed with the gene

S100A7 whose expression was highly downregulated in the latter, especially compared to the

tumour (Figure 3.20). Whilst S100A7 expression decreased slightly in UW-CSCC1 compared to the originating tumour, complete loss of expression can be seen in UW-CSCC1-R.

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Tumour UW-CSCC1 UW-CSCC1-R

0 5 10 15 Log 2 c o u n ts

Figure 3.20 Log2 counts of S100A7 between PDCCs and the matched tumour.

This gene encodes for psoriasin which is overexpressed in skin wounds. Psoriasin is regarded as having cell adhesion properties; ergo the loss of gene expression in UW-CSCC1-R aligns with observations of other cell adhesion genes.

Several genes relating to ECM structure were upregulated in UW-CSCC1-R, notably

PCOLCE. This gene codes for a glycoprotein that drives collagen degradation, thus helping to

explain the more invasive response observed with UW-CSCC1-R in the organotypic assay presented in Chapter 2, section 2.3.6.

3.4.5.2 DNA damage repair

DNA damage-repair mechanisms were significantly upregulated in UW-CSCC1-R. This may explain how this cell line came to survive irradiation in the first place. It is theorised that a subpopulation of cells in UW-CSCC1 possessed elevated DNA damage-repair activity and when exposed to irradiation, they were able to utilise this characteristic to survive. Of note,

PCNA, MAD2L2, and FEN1 were upregulated in UW-CSCC1-R and contribute major roles in

mediating the DNA damage repair required for the survival of UW-CSCC1-R (Gomes and Burgers, 2000; Boersma et al., 2015; Boehm et al., 2016). Further efforts are required to dissect the role these DNA damage repair genes play specifically in UW-CSCC1-R. For example, knock-down of components of this pathway may result in apoptosis or confer greater sensitivity towards additional radiation.

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3.4.5.3 Stemness

Cancer stem cells have been well described in the literature for many other malignancies following radiation exposure, including squamous skin tumors (Driessens et al., 2012), colon cancer (Sahlberg et al., 2014), and breast cancer (Phillips et al., 2006). These populations have all demonstrated a faster population doubling time following treatment, like with UW- CSCC1-R, and likely contribute to the recurrence and rapid spread of disease.

One of the most upregulated genes in UW-CSCC1-R was HAPLN1, which has been documented as a playing a major role in a signaling network which leads to stemness and mesenchymal properties in hepatocellular carcinomas (Mebarki et al., 2016). The upregulation in UW-CSCC1-R of additional genes generally specific to stem cells, namely

SOX2, CD24, and PROM1 (CD133), further add to this theory of increased stemness

following radiation therapy.

3.4.5.4 Cytoskeletal organisation

Genes encoding for keratin family proteins, KRT7, KRT14, and KRT19 were downregulated in UW-CSCC1-R. These genes encode for proteins that are crucial to cytoskeletal formation in keratinocytes. Therefore, the downregulation of these genes relates to the degree of differentiation, aligning with morphological observations of the PDCCs (Chapter 2).

3.4.6 Conclusions and future directions

Whilst the establishment of the PDCCs are a significant contribution in the journey towards understanding metastatic cSCC, they should not be considered as unique, authoritative models. Numerous differences between the PDCCs and their clinical counterpart were identified, although the impact of these may be insignificant for most relevant cancer research purposes, i.e. investigating drug sensitivity. Moving forward with these cell lines, one must consider the assay being used and its compatibility with the genomic and transcriptomic changes the PDCCs have incurred. Weinstein, (2012) reflected on the words of statistician

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George Box, “all models are wrong, but some are useful”, a sentiment particularly meaningful

in cell culture. The novel PDCCs described in this chapter represent the starting pieces of the puzzle; whilst far from complete, the first steps have been taken to allow others to fall into place – the potential value of which cannot be understated.

The fact that the majority of pathways are restored in the xenograft demonstrated that the PDCCs are at the very least an economic method of preserving genomic information for eventual restoration in an animal model.

Moving forward, animal models with the UW-CSCC series could be developed to validate in

vitro observations of molecular targets for therapy, prognostic factors, and therapeutic

responsiveness to novel and established agents (in association with chapter 5).

As far as 2D cell models go, UW-CSCC1 is just as good, if not better than many other cancer models for HTS drug screening. However, given the differences between the cell lines and the clinical tumour, it is obvious that cell lines are an unreliable platform for biomarker discovery and to a lesser extent, drug target discovery. As such, clinical specimens are required to discover candidate biomarkers/drug targets for cSCC, an undertaking presented in Chapter 4 of this thesis.

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CHAPTER 4: COMPARATIVE ANALYSES OF CLINICAL