Spatial expression of IKK-alpha is associated with a differential mutational landscape and survival in primary colorectal cancer

Abstract

Background: To understand the relationship between key non-canonical NF-κB kinase IKK-alpha(α), tumour mutational profile and survival in primary colorectal cancer.

Methods: Immunohistochemical expression of IKKα was assessed in a cohort of 1030 patients who had undergone surgery for colorectal cancer using immunohistochemistry. Mutational tumour profile was examined using a customised gene panel. Immunofluorescence was used to identify the cellular location of punctate IKKα expression.

Results: Two patterns of IKKα expression were observed; firstly, in the tumour cell cytoplasm and secondly as discrete 'punctate' areas in a juxtanuclear position. Although cytoplasmic expression of IKKα was not associated with survival, high 'punctate' IKKα expression was associated with significantly reduced cancer-specific survival on multivariate analysis. High punctate expression of IKKα was associated with mutations in KRAS and PDGFRA. Dual immunofluorescence suggested punctate IKKα expression was co-located with the Golgi apparatus.

Conclusions: These results suggest the spatial expression of IKKα is a potential biomarker in colorectal cancer. This is associated with a differential mutational profile highlighting possible distinct signalling roles for IKKα in the context of colorectal cancer as well as potential implications for future treatment strategies using IKKα inhibitors.

Fig. 1. Representative images of IKKα immunohistochemical staining and controls.

Example images of weak (a) and strong (b) cytoplasmic staining of IKKα. Representative images of true negative pancreatic tissue (c) and true positive kidney tissue (d) staining, respectively. Negative control colorectal tissue (e) with no antibody added during IHC staining process and colorectal tissue used as a positive control identified during optimisation (f). Western blot probed for IKKα and IKKβ using IKKα and IKKβ silenced lysates, MCF-7 cell pellets (IKKα silenced/IKKβ silenced) stained for IKKα as shown previously by Bennett et al. [3] (g). The optimal threshold for cytoplasmic IKKα histoscore was determined using R (h).

Fig. 2. Representative images of punctate IKKα immunohistochemical staining and controls.

Example images of low (a) and high (b) punctate staining of IKKα.

Fig. 3. Flow diagram demonstrating patient exclusions.

Patients with missing cores or insufficient tumour for analysis were excluded. Thereafter patients who had either died within 30 days of surgery, had inflammatory bowel disease-related malignancy or had received neoadjuvant chemotherapy were excluded from final analysis.

Fig. 4. IKKα expression and survival.

Expression of IKKα in the cytoplasm was not associated with CSS in the full cohort (a). High expression of cytoplasmic IKKα was associated with significantly worse cancer-specific survival in patients with right-sided tumours (b) but not in those with left-sided or rectal tumours (c, d). Punctate expression of IKKα was associated with CSS in the full cohort (e).

Fig. 5. Mutational analysis.

Oncoplots (a–c) and forest plots (d) demonstrating the top 10 mutated genes in low and high cytoplasmic IKKα. Oncoplots (e–g) and forest plots (h) demonstrating the top 10 mutated genes in low and high punctate IKKα.

Fig. 6. IKKα dual fluorescence with Rab5, Rab7 and Golgi 58.

Co-localisation between IKKα (red) and Rab5 and Rab7 (green) was not observed in colorectal cancer tissue. DAPI (blue) represents nuclear counterstain. IKKα co-located with a Golgi marker (green) in colorectal cancer tissue.