Loss of Myosin Vb in colorectal cancer is a strong prognostic factor for disease recurrence

Figures

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  Figure 1. Bioinformatic analysis reveals the potential use of MYO5B as a diagnostic marker for CRC. (A) Dot plot showing the log2 FC values of the Myosin V family genes in adenoma (beige) and CRC (blue) samples compared to healthy colorectal mucosa samples (grey) in a meta-analysis of different CRC data sets, including 829 patients. Bar represents the mean expression intensity (***P<0.001, t-test corrected for multiple testing). (B) MYO5B expression according to the different TNM stages in the meta-analysis. Bar represents the mean expression intensity (*P<0.05, ***P<0.001, t-test corrected for multiple testing). (C) ROC curves with corresponding AUC values for MYO5A, MYO5B, and MYO5C when comparing CRC and healthy samples in the meta-analysis. Distribution of gene expression values for healthy and CRC samples are shown in the insets.

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  Figure 2. Downregulation of MYO5B on mRNA and protein levels in human CRC. (A) MYO5B mRNA levels in bulk tissue of matched tumour (CRC, n=50) and non-tumour (normal, n=50) samples from CRC patients. Data are presented as mean±s.d. (***P<0.001, paired t-test). (B) MYO5B mRNA levels in laser-microdissected tumour (CRC, n=67) and non-tumour (normal, n=21) samples. Data are presented as mean±s.d. (***P<0.001, unpaired t-test). (C) In 20 out of 21 paired microdissected tumour samples, MYO5B mRNA levels were higher in non-tumour tissue compared to the matched tumour sample (***P<0.001, paired t-test). (D) Immunohistochemical staining of 74 paired TMAs of CRC patients. Left panel: representative image of MYO5B immunostaining on a section that contains normal (bottom) and cancerous tissue (top). Staining was mainly cytoplasmic and showed the expected accumulation of MYO5B at the apical brush border of normal enterocytes, as well as in tumoural cells in which differentiation was still preserved. Scale bar corresponds to 200 μm. Right panel: quantification of MYO5B staining of matched tumour (CRC; n=74) and non-tumour (normal; n=74) samples scored according to the staining intensity of…

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  Figure 3. Frequently reduced expression of MYO5B is neither due to methylation nor mutations in CRC. (A) 5-aza-dC (DAC) treatment does not increase basal MYO5B expression in colon cancer cell lines. Data are representative of 2–4 independent experiments and presented as mean±s.d. (B) Methylation pattern analysed by mass array technology of eight CRC cell lines as well as eight tumour samples and four matching non-tumour counterparts. Each analysed cytosine is represented by a dot; cytosines with values below 5% were considered as non-methylated. (C) Analysis of MYO5B methylation in the TCGA data set covering methylation data on 295 tumour and 19 non-tumour samples. (D) MYO5B mutations in CRC patients. The protein domains and motifs are presented according to Qiu et al (2017), and the RAB8A- and RAB11A-binding sites according to Roland et al (2011). The dilute domain is also called globular tail domain. IQ=IQ motif, BD=binding domain.

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  Figure 4. MYO5B is a prognostic marker in CRC patients. (A and B) Overall (A), n=585 and relapse-free (B), n=574 survival in CRC patients stratified according to MYO5B expression in the GSE39582 data set. (C and D) Overall (C) and relapse-free (D) survival in early stage (stages I and II) CRC patients stratified according to MYO5B expression in the GSE39582 data set. (E) Disease-free survival curves for patients with ‘high’ and ‘low’ expression levels of MYO5B in an independent data set (GSE24551) covering 160 patients. (F) Metastasis-free survival based on ‘high’ or ‘low’ expression of MYO5B in a data set (GSE28814) containing clinical data on metastasis from 125 CRC patients. Patient numbers and associated P-values are indicated in the figures.

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  Figure 5. RAB family members RAB8A and RAB25 are downregulated in CRC. (A) Dot plot showing the log2 expression intensities of different RAB genes in adenoma (beige) and CRC (blue) samples compared to unmatched healthy colorectal mucosa (grey) samples in a meta-analysis of different CRC data sets, including 829 patients. Bar represents the mean expression intensity (*P<0.05, ***P<0.001, t-test corrected for multiple testing). (B) ROC curves with corresponding AUC values for RAB genes when classifying CRC and healthy patients in the meta-analysis. Distribution of gene expression values for normal and CRC samples are shown in the insets. (C) RAB8A and RAB25 expression levels in bulk tissue of matched tumour (CRC, n=50) and non-tumour (normal, n=50) samples from CRC patients. Data are presented as mean±s.d.; *P<0.05, ***P<0.001, paired t-test.

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  Figure 6. The combination of MYO5B expression with its adapter protein RAB8A improves the prognostic power of MYO5B in CRC. (A) Top 20 significant combinatorial signatures between MYO5B and different genes of the RAB family for overall (OS, left) and relapse-free (RFS, right) survival in 585 CRC patients of the GSE39582 data set. Because of space issues, MYO as well as RAB have been omitted from the name description and only the last letters are shown. (B) Overall (left) and relapse-free (right) survival in 585 CRC patients stratified according to their combined RAB8A and MYO5B expression signature in the GSE39582 data set. The dotted line represents the MYO5B signature alone, whereas continuous lines represent the combinatorial signature. Patient numbers as well as their P-values are indicated in the figure.