AKT3 Expression in Mesenchymal Colorectal Cancer Cells Drives Growth and Is Associated with Epithelial-Mesenchymal Transition

Abstract

Colorectal cancer (CRC) is a heterogeneous disease that can currently be subdivided into four distinct consensus molecular subtypes (CMS) based on gene expression profiling. The CMS4 subtype is marked by high expression of mesenchymal genes and is associated with a worse overall prognosis compared to other CMSs. Importantly, this subtype responds poorly to the standard therapies currently used to treat CRC. We set out to explore what regulatory signalling networks underlie the CMS4 phenotype of cancer cells, specifically, by analysing which kinases were more highly expressed in this subtype compared to others. We found AKT3 to be expressed in the cancer cell epithelium of CRC specimens, patient derived xenograft (PDX) models and in (primary) cell cultures representing CMS4. Importantly, chemical inhibition or knockout of this gene hampers outgrowth of this subtype, as AKT3 controls expression of the cell cycle regulator p27^KIP1. Furthermore, high AKT3 expression was associated with high expression of epithelial-mesenchymal transition (EMT) genes, and this observation could be expanded to cell lines representing other carcinoma types. More importantly, this association allowed for the identification of CRC patients with a high propensity to metastasise and an associated poor prognosis. High AKT3 expression in the tumour epithelial compartment may thus be used as a surrogate marker for EMT and may allow for a selection of CRC patients that could benefit from AKT3-targeted therapy.

Keywords: AKT3; CMS; growth; mesenchymal CRC.

Figure 1

Identification of differentially expressed kinases in CMS4 versus CMS1-3 samples. (a,b) Heatmaps depicting Z-score transformed 2Log expression of kinases significantly differentially expressed in CRC patient samples (a) and (primary) cell lines (b); (c,d) 2Log mRNA expression levels of kinases differentially expressed between subtypes in both patient samples (c) and (primary) cell lines (d) as determined by microarray or RNAseq.

Figure 2

AKT3 is more highly expressed in CMS4 human CRC cell lines and tumours. (a) Quantitative real time PCR analysis on 29 established CRC cancer cell lines for AKT3 expression. Expression level was normalised to ATP5E expression, average normalised expression of N = 3, each performed with technical triplicates, is plotted per cell line. Normalisation to GUSB yielded similar results; (b) Western blot analysis for AKT3 expression in the same cell line panel as in a. The CMS4 cell line Colo320-HSR was included on every blot as a positive control. Numbers on right hand side of blots indicate position of specified protein molecular weight marker. 2,2,2-Trichloroethanol (2,2,2TCE) signal (excerpt taken around 60 kDa region) indicates amount of protein loaded per cell line; (c) 2Log expression of AKT3 in the murine stroma and human cancer cells in PDX models as determined by RNAseq; (d) Representative images of RNAscope staining for AKT3 (brown dots) on human CRC tissue slides. Sections were counterstained with haematoxylin. AKT3 expression was only detected in the stroma of Co454, whereas the epithelium of Co447 also stained positive. MSI, microsatellite instable; MSS, microsatellite stable. Scale bars 50 µm.

Figure 3

AKT3 is activated in and contributes to growth of CMS4 cell lines. (a) Images of western blots performed on AKT3 IP samples of HT55 (CMS2), Colo320-HSR and Hutu-80 (CMS4). Two different exposures are shown for the anti-AKT3 western blots (short = middle panel) and long (bottom panel). Of note IP lysates of each cell line were equally divided over two lanes and then run and blotted on the same gel and membrane. Membrane was cut into two and parts were either probed for serine 473 phosphorylated AKT or AKT3. Signal at the bottom of blots is specific signal coming from the IgG heavy chain. Representative blots of one experiment are shown (total N ≥ 3); (b,d) Western blot for phospho-AKT (Ser473) in cells treated with 1.25 µM of indicated inhibitor. Numbers at bottom of blots indicate phospho-AKT signal relative to control condition, as calculated after densitometric analysis of bands; (c,e) Bar graphs depicting the average decrease in CellTiter Blue signal of CMS2 (c) and CMS4 (e) cell lines upon treatment with AKT1/2 kinase inhibitor or AKT1/2/3 inhibitor (MK2206). All values were normalised to signal in DMSO treated control conditions. Bar graphs indicate mean of three independent experiments, error bars indicate S.D.; (f) Bar graphs depict outgrowth of polyclonal WT population and AKT3 KO clones relative to initial number of cells plated. One representative experiment is shown for 3 independent experiments. Error bars represent S.D. p-values calculated using two-tailed Welch’s t-test. * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001; (g) Western blot analysis for p27^KIP1 expression in polyclonal WT population versus AKT3 KO clones. Blots shown are representative for 2 independent experiments. Numbers at bottom of blots indicate p27^KIP1 signal relative to control condition, as calculated after densitometric analysis of bands. Numbers on right hand side of all blots in this figure indicate position of specified protein molecular weight marker.

Figure 4

High AKT3 and FRMD6 expression levels are linked to EMT gene set enrichment and relapse-free survival. (a,c) Gene set enrichment analysis (GSEA) on 20% high versus 20% low AKT3 expressing CRC cell lines (a) and CMS4 CRC cell lines (c); (b) Heatmap depicting Z-score transformed 2Log expression levels of EMT genes in CRC cell lines. CMS1: orange, CMS2: blue, CMS3: pink, CMS4: green, unclassified: white. 2Log AKT3 expression levels are indicated on top of heatmap in green gradient; (d) GSEA on 50% high versus 50% low AKT3 expressing carcinoma cell lines included in the CCLE panel; (e) Heatmap depicting Z-score transformed 2Log expression levels of EMT genes in panel of CCLE carcinoma cell lines. Colours on top indicate carcinoma of origin; biliary tract: grey, breast: red, endometrium: pink, large intestine: yellow, oesophagus: brown, pancreas: purple, prostate: blue, small intestine: orange, stomach: green. 2Log AKT3 expression levels are indicated on top of heatmap in green gradient; (f) Kaplan-Meier curves for relapse-free survival of 376 MSS stage I-III CRC patients present in the CMS consortium dataset, for whom clinical outcome data is publicly available. Last quartile (top 25%) of patients most highly expressing AKT3 were compared rest of patients in dataset. P-value calculated using Mantel-Cox test; (g) Scatter plot depicts 2Log expression of AKT3 and FRMD6 in each sample of CCLE CRC cell line panel. R- and p-value calculated using two-tailed Pearson correlation; (h) GSEA on 20% high versus 20% low FRMD6 expressing CRC cell lines; (i) Kaplan-Meier curves for relapse-free survival on same patient dataset as in (f). Last quartile (top 25%) of patients most highly expressing FRMD6 were compared to rest of patients in dataset. p-value calculated using Mantel-Cox test.