High CLU expression has been associated with various cancers, including CRC [
61]. Moreover, the abundance, intracellular localisation and histological distribution of CLU expression have been associated with the progression of CRC [
38,
62‐
65] (Table
1). Conversely, a recent pan-cancer analysis by Fu and colleagues of
CLU expression revealed decreased expression across most cancers compared to the matched normal tissue, including CRC [
66]. However, this could be attributed to the opposing functions of the CLU isoforms which as previously mentioned, play very different roles in the cell. For example, elevated expression of sCLU has been reported in invasive colorectal adenocarcinomas, whereas expression in normal colonic tissues was detected only at very low levels [
64]. Similar observations across different stages of colorectal tumorigenesis have also been reported, with 17% of the adenomas, 46% of the primary CRCs and 57% of the CRC metastatic lesions displaying overexpression of cytoplasmic CLU, compared to normal mucosa [
62]. Whilst upregulation of CLU can occur in the early stages of premalignant adenomatous polyp formation, overexpression of CLU has been significantly correlated with advanced clinical stage [
62]. High CLU expression also correlated with poor outcomes in stage II CRC within a cohort of 202 patients [
63]. Additionally, sCLU was found to be abundant in the epithelium of tumour tissue, whereas in normal mucosa, sCLU is more abundant in the stroma [
63]. Conversely, expression of nCLU was found to be decreased in colon cancer tissues compared to matched normal control tissues [
67]. Whilst the nCLU isoform is pro-apoptotic and primarily expressed in normal colon epithelium, during tumour progression, expression of nCLU decreases with more sCLU translocated into the cytoplasm where it plays a protective role in preventing apoptosis [
27,
68]. This shift in the expression of CLU isoforms is linked to increased tumour cell survival, aggressiveness and enhanced metastatic potential [
69]. Therefore, these studies highlight an oncogenic role of sCLU during tumour progression and its potential as a diagnostic biomarker.
Table 1
Expression of clusterin (CLU) across normal, adenomatous and tumour tissue of colorectal origin
| Normal colonic tissue | / | − | − (very weak) | 2 |
Hyperplastic polyps | / | − | + | 3 |
Tubular adenomas | / | + + + | + + + | 1 |
Villous adenomas | / | + + + | + + + | 1 |
Invasive adenocarcinomas | n/a | − | + + | 1 |
| Normal colonic tissue | / | + | − | 30 |
Adenoma | / | + | + | 10 |
Adenocarcinoma | I to II | − | + + + | 10 |
Adenocarcinoma | III to IV | − | + + + + | 10 |
| Normal colonic mucosa | / | n/a | + (100%) | 76 |
Adenoma | / | n/a | + + + (17%) | 20 |
Primary carcinoma | II | n/a | + + + (33%) | 42 |
Primary carcinoma | III-IV | n/a | + + + (60%) | 43 |
Metastases | IV | n/a | + + + (57%) | 35 |
| | | | Epithelium | Stroma | |
Normal colonic mucosa | / | n/a | + | + + | 202 |
Colorectal cancer | II | n/a | + + + | + | 202 |
3.1 Clusterin expression as a diagnostic marker
Early-stage CRC is often treatable through surgical interventions and as such, early detection through the implementation of screening programs such as the faecal occult blood test has significantly reduced the burden of disease [
70].
CLU expression has shown promise as a predictive biomarker for the identification of individuals at risk of developing CRC or in the early stages of disease [
64,
71‐
74]. The progressive increase of sCLU expression in the setting of CRC correlates with a significant increase of CLU in the serum and stool of CRC patients [
27,
73]. Moreover, Mazzarelli et al. [
27] identified a significant positive correlation between CLU expression in stool and more advanced stage of disease [
27]. In animal studies, it was also demonstrated that CLU secreted from a colon cancer cell line (Caco-2) injected into mice was detectable in blood samples, with the increasing level of CLU correlating with the increasing dimension of the tumours [
72]. A positive correlation was identified between
CLU expression and tumour severity with elevated expression of CLU also associated with a decrease in disease-free survival in CRC [
24,
63,
65,
75] (Fig.
1). Again, Fu et al. [
66] noted conflicting results from their pan-cancer analysis regarding
CLU expression and overall survival, with high
CLU expression conferring a survival advantage in some cancers, and a disadvantage to others (including CRC). Therefore, this suggests that monitoring expression levels of the sCLU isoform specifically may be a useful biomarker for the detection of disease as well as potential as a surveillance tool, in a similar approach to monitoring of circulating tumour DNA for the detection of disease relapse.
3.2 Clusterin in chemoresistance and metastasis
In addition to its use as a diagnostic marker, high CLU expression is also associated with advanced tumours which are more prone to resist chemotherapy treatment and metastasis. Upregulated expression of CLU has been linked to increased chemoresistance in multiple cancer types including the breast, lung, prostate, bladder, liver, pancreatic, ovarian, cervical, melanoma and osteosarcoma [
26,
38]. As the sCLU isoform has an anti-apoptotic function, the suppression of CLU expression may promote cell death when challenged by chemotherapy. Studies which modulate CLU expression have found that decreasing endogenous CLU expression through the means of drug [
50,
53,
76], antisense oligonucleotide [
77‐
81] or siRNA inhibition [
82,
83] increases sensitivity to chemotherapeutics and reduces overall tumour burden (as reviewed in detail by Praharaj et al. [
38]). In contrast, treatment with exogenous CLU results in increased resistance to chemotherapeutics [
84], despite the fact that sCLU in conditioned media failing to demonstrate an anti-apoptotic effect in cell lines [
42]. Combination therapy involving an antisense oligonucleotide, Custiren (OSX-011, an inhibitor of sCLU) [
77,
79] with chemotherapeutics in various cancers, including prostate [
85,
86], lung [
87] and breast cancer [
88] demonstrated improved patient survival in Phase II clinical trials. However, during Phase III synergy trials, OGX-011 combined with prednisone and cabazitaxel [
89] or docetaxel [
90] showed no significant improvement in overall survival in castration-resistant prostate cancer patient.
CLU expression has recently been examined in CRC patient-derived organoids (PDOs) that had been treated with the chemotherapeutic 5-FU [
24].
CLU expression is not only significantly increased after 5 days of chemotherapy treatment
in vitro but also correlated with an increase in PDO resistance to chemotherapy [
24]. In hepatocellular carcinoma, sCLU was found to induce resistance to the chemotherapeutic oxaliplatin via activation of the phosphoinositide-3-kinase–protein kinase B (PI3K)/Akt pathway [
91] (Fig.
1). Further analysis revealed that sCLU regulates PI3K/Akt pathway via downregulation of growth arrest and DNA-damage-inducible 45 alpha (Gadd45a) which itself decreases phosphorylation of Akt [
92] (Fig.
1). Another study also explored the relationship between CLU expression and chemoresistance specifically in CRC by generating a SW480 CRC cell line that overexpresses intracellular sCLU. Interestingly, the sCLU overexpressing cells were more sensitive to combined chemotherapy treatment under normal and hypoxic conditions [
93]. Therefore, further exploration of the role of CLU in therapy resistance in CRC is warranted, with consideration of factors including different cancer stages, mutation profiles and consensus molecular subtypes (CMSs) required.
In addition to chemoresistance, an increase in
CLU expression has also been linked to metastasis. Flanagan et al. [
25] generated a breast cancer cell line which overexpressed sCLU to explore the effect on treatment response. This study found that sCLU-overexpressing cells transplanted into host mice were more resistant to cytokine-induced apoptosis and were also more likely to metastasise to the lung compared to the parental cell line [
25]. Moreover, upregulation of
CLU expression combined with L1CAM mediated signalling, a marker of tumour cells with metastatic potential [
94], within LS174T CRC tumour cells results in substantial metastasis formation within the liver and spleen after transplantation (Fig.
2) [
28]. Subsequent suppression of
CLU expression via shRNA significantly decreased the number and size of these metastases [
28]. Similarly, silencing
CLU expression via siRNA in SW480, SW620, and Caco2 CRC cell lines
in vitro reduced their proliferative and migratory capabilities [
95].
Furthermore, it was shown that
CLU expression is necessary for TGFβ-induced cell migration as knockdown of both
CLU, and its transcriptional regulator
Twist1, significantly reduced the number of invasive prostate cancer cells [
60] (Figs.
1,
2). This suggests that CLU may mediate epithelial-mesenchymal transition and thus metastasis formation through activation of the TGFβ signalling pathway. In contrast, overexpression of
MEG3 in CRC cell lines significantly inhibited cell proliferation and cell migration
in vitro which corresponded to a reduction in tumour growth and metastasis formation in xenograft models [
57].