LRP1 expression in colon cancer predicts clinical outcome

Abstract

LRP1 (low-density lipoprotein receptor-related protein 1), a multifunctional endocytic receptor, has recently been identified as a hub within a biomarker network for multi-cancer clinical outcome prediction. As its role in colon cancer has not yet been characterized, we here investigate the relationship between LRP1 and outcome.

Materials and methods: LRP1 mRNA expression was determined in colon adenocarcinoma and paired colon mucosa samples, as well as in stromal and tumor cells obtained after laser capture microdissection. Clinical potential was further investigated by immunohistochemistry in a population-based colon cancer series (n = 307). LRP1 methylation, mutation and miR-205 expression were evaluated and compared with LRP1 expression levels.

Results: LRP1 mRNA levels were significantly lower in colon adenocarcinoma cells compared with colon mucosa and stromal cells obtained after laser capture microdissection. Low LRP1 immunohistochemical expression in adenocarcinomas was associated with higher age, right-sided tumor, loss of CDX2 expression, Annexin A10 expression, CIMP-H, MSI-H and BRAFV600E mutation. Low LRP1 expression correlated with poor clinical outcome, especially in stage IV patients. While LRP1 expression was downregulated by LRP1 mutation, LRP1 promoter was never methylated.

Conclusions: Loss of LRP1 expression is associated with worse colon cancer outcomes. Mechanistically, LRP1 mutation modulates LRP1 expression.

Keywords: BRAF; LRP1; Pathology; colorectal cancer; miR-205; microsatellite instability.

Figure 1. LRP1 expression in colon cancer cells compared to normal colon and stromal cells

(A) qRT-PCR expression levels of LRP1 mRNA in colon adenocarcinoma fresh frozen samples compared with normal colon mucosa fresh frozen samples. Values are shown as dCt normalized with RPL32 (B) Comparative quantification analysis of LRP1 mRNA expression levels in tumor samples compared with paired normal colon mucosa samples. Values are shown as ddCt fold induction. ^****p < 0.0001, Mann Whitney test. (C–H) Representative microphotographs of LRP1 immunohistochemistry on colon mucosa (C–D) and colon adenocarcinoma (E–H). (C) LRP1 expression in surface epithelium (arrows) in normal colon mucosa (×5 magnification). (D) LRP1 expression in fibroblasts of the lamina propria (arrows) in normal colon mucosa (×10 magnification). Loss of LRP1 expression in malignant cells of a moderately differentiated adenocarcinoma (E) and a mucinous adenocarcinoma (F) (×20 magnification). (G) Loss of LRP1 expression in malignant cells (asterisks) and stromal lymphocytes (arrows) of a poorly differentiated adenocarcinoma (×30 magnification). (H) LRP1 expression in malignant and stromal cells of a moderately differentiated adenocarcinoma (×20 magnification).

Figure 2. Laser capture microdissection analyses

(A–B) Representative microphotographs of microscopic control of laser capture microdissection (LCM) (Cresyl violet, ×20 magnification). (A) Microdissection of the malignant cells. (B) Microdissection of the stromal cells. Residual malignant glands are highlighted with an asterisk. (C) qRT-PCR expression levels of CEA mRNA in adenocarcinoma cells compared with stromal cells after LCM. Values are shown as dCt normalized with RPL32. (D) Comparative quantification analysis of CEA mRNA expression levels in tumor cells compared with stromal cells after LCM. Values are shown as ddCt fold induction. (E) qRT-PCR expression levels of LRP1 mRNA in adenocarcinoma cells compared with stromal cells after LCM. Values are shown as dCt normalized with RPL32. (F) Comparative quantification analysis of LRP1 mRNA expression levels in tumor cells compared with stromal cells after LCM. Values are shown as ddCt fold induction. ^*p < 0.05; ^**p < 0.01; ^****p < 0.0001, Mann Whitney test. (G) Linear regression analysis of LRP1 mRNA expression levels evaluated by qRT-PCR on complete fresh frozen adenocarcinoma sample against LRP1 IHC score of tumor cells obtained by multiplying staining intensity (0 to 3) and percentage of positive cells (0 to 4). (H) Linear regression analysis of LRP1 mRNA expression of tumor cells against LRP1 IHC score of tumor cells obtained by multiplying staining intensity (0 to 3) and percentage of positive cells (0 to 4) after LCM.

Figure 3. Correlation of LRP1 mRNA levels with clinical and molecular findings

Left panel: LRP1 mRNA levels analyses by qRT-PCR (dCt normalized with RPL32) on fresh frozen colon adenocarcinoma samples from our cohort compared with age (A), BRAFV600E mutation (B) and CpG island methylator phenotype (CIMP-H) (C). Right panel: Correlation analysis of LRP1 mRNA expression levels extracted from the colorectal cancer cohort of the TCGA, as retrieved using cBioportal for Cancer Genomics (http://cbioportal.org) web resources with sided adenocarcinomas (D), BRAF mutation (E), CIMP status (F), MSI status (G). and CDX2 mRNA expression (H). ^*p < 0.05; ^**p < 0.01; ^***p < 0.001; ^****p < 0.0001, Mann Whitney test. Abbreviations: H, high; L, Low; MSI, microsatellite instability; MSS, microsatellite stability; CIMP, CpG island methylator phenotype.

Figure 4. Survival analysis in colon cancer patients from our cohort compared with LRP1 immunohistochemical expression in tumor cells

Kaplan-Meier curves of overall survival and event or progression free-survival probability for low (red line) and high (blue line) LRP1 immunohistochemical (IHC) score in adenocarcinoma cells whatever the tumor stage (A, B), in stage IV (metastatic) patients (C, D) and in stage IV patients treated with bevacizumab (E, F). IHC score were evaluated by multiplying staining intensity (0 to 3) and percentage of positive malignant cells (0 to 4) obtained with anti-LRP1 clone 8G1 immunolabelling. Median IHC score was used to separate low (score 0 to 4) and high (score 6 to 12) LRP1 IHC score. All p values were calculated using the log rank test.

Figure 5. Event-free survival analyses in an independant cohort

Publicly available SieberSmith gene expression dataset was obtained from R2 microarray analysis and visualization platform (http://r2.amc.nl), and used for survival analyses. Event-free survival Kaplan-Meier curves for LRP1 mRNA expression in all stages (A), in stage II (B) and in stage III patients (C). (D) Progression-free survival Kaplan-Meier curve for LRP1 mRNA expression in stage IV patients. All p values were calculated using the log rank test and computed using R2 online tools.

Figure 6. Analysis of LRP1 expression regulation by LRP1 gene mutation or methylation

(A) Somatic mutation data from the complete length of LRP1 gene obtained from colorectal cancer of the TCGA cohort using cBioportal for Cancer Genomics (http://cbioportal.org) web resources. Colored boxes present on the LRP1 gene representation correspond to exons encoding functional domains of LRP1. Green domain, low-density lipoprotein receptor domains; blue, low-density lipoprotein receptor repeats; yellow, coagulation factor Xa inhibitory site; orange, domain of unknown function; red, calcium-binding EGF domain; violet, complement Clr-like EGF-like. (B) Graphical representation of association of LRP1 mutational status with clinical and molecular tracks and LRP1 mRNA expression. (C) LRP1 mRNA expression levels comparison between LRP1 mutated and LRP1 wild type colorectal cancer. ^***p = 0.003, Mann Whitney test. Linear regression analyses between LRP1 mRNA expression levels and promoter methylation (D), intronic methylation (E), global DNA methylation levels approximated by LINE1 (F) in our cohort. (G) Correlation of LRP1 mRNA expression levels and LRP1 promoter methylation in data extracted from the TCGA.

Figure 7. Comparison of miR-205 and miR-338-5p expression with LRP1 expression

Analyses of miR-205 (A) and miR-338-5p (B) expression by qRT-PCR in fresh frozen colon cancer adenocarcinoma compared with normal colon mucosa from our cohort (^*p < 0.05, ^***p < 0.001, Mann Whitney test). Linear regression analysis of LRP1 mRNA expression levels evaluated by qRT-PCR on complete fresh frozen adenocarcinoma sample against miR-205 (C) and miR-338-5p (E) expression. Linear regression analysis of miR-205 (D) and miR-338-5p (F) expression against LRP1 immunohistochemical (IHC) score of tumor cells. IHC score was assessed by multiplying staining intensity (0 to 3) and percentage of positive tumor cells (0 to 4) with anti-LRP1 clone 8G1 immunolabelling.