KRAS mutation-independent downregulation of MAPK/PI3K signaling in colorectal cancer

Abstract

KRAS is a gatekeeper gene in human colorectal tumorigenesis. KRAS is 'undruggable'; hence, efforts have been diverted to inhibit downstream RAF/MEK/ERK and PI3K/Akt signaling. Nevertheless, none of these inhibitors has progressed to clinical use despite extensive trials. We examined levels of phospho-ERK1/2(T202/Y204) and phospho-Akt1/2/3(S473) in human colorectal tumor compared to matched mucosa with semi-quantitative near-infrared western blot and confocal fluorescence immunohistochemistry imaging. Surprisingly, 75.5% (25/33) of tumors had lower or equivalent phospho-ERK1/2 and 96.9% (31/32) of tumors had lower phospho-Akt1/2/3 compared to matched mucosa, irrespective of KRAS mutation status. In contrast, we discovered KRAS-dependent SOX9 upregulation in 28 of the 31 (90.3%) tumors. These observations were substantiated by analysis of the public domain transcriptomics The Cancer Genome Atlas (TCGA) and NCBI Gene Expression Omnibus (GEO) datasets and proteomics Clinical Proteomic Tumor Analysis Consortium (CPTAC) dataset. These data suggest that RAF/MEK/ERK and PI3K/Akt signaling are unlikely to be activated in most human colorectal cancer.

Keywords: CPTAC; KRAS signaling; MAPK/PI3K; SOX9; TCGA; colorectal tumorigenesis.

Fig. 1

Downregulation of phospho‐ERK1/2 in CRC tumor compared to matched mucosa. A representative NI‐WB of phospho‐ERK1/2 and total‐ERK1/2 for 7 pairs of CRC tumor and matched mucosa with tumor KRAS mutation status indicated on top (A). Box and whiskers plot of NI‐WB phospho‐ERK/total‐ERK I.I K counts ratio for ERK1 and ERK2 of pooled mucosa and tumor (B), of tumor grouped by KRAS mutation (C), Duke’s stage (A/B early stage; C/D advanced stage) (D) and tumor site (right‐colon; left‐colon) (E). Representative confocal images of phospho‐ERK1/2 (green) IHC staining with nuclear DAPI (blue) counterstain of CRC‐matched mucosa and tumor cryosections for each KRAS mutation status, scale bar = 50 µm in top left image is applicable to all images (F). *two‐tailed t‐test P < 0.05; i, paired t‐test; ii, unpaired t‐test with Welch’s correction. M, mucosa; p‐ERK1/2, phospho‐ERK1/2; T, tumor; tERK, total‐ERK. Both NI‐WB (A‐D) and IHC (F) were performed on biological replicates (n = 33 and n = 8, respectively).

Fig. 2

Downregulation of phospho‐Akt1/2/3 in CRC tumor compared to matched mucosa. A representative NI‐WB of phospho‐Akt1/2/3 and pan‐Akt for 7 pairs of CRC tumor and matched mucosa with tumor KRAS mutation status indicated on top (A). Box and whiskers plot of NI‐WB phospho‐Akt1/2/3 / pan‐Akt I.I K counts ratio of pooled mucosa and tumor (B), of tumor grouped by KRAS mutation (C), Duke’s stage (A/B early stage; C/D advanced stage) (D) and tumor site (right‐colon; left‐colon) (E). *two‐tailed t‐test P < 0.05; ****two‐tailed t‐test P < 0.0001; i, paired t‐test; ii, unpaired t‐test with Welch’s correction. M, mucosa; p‐Akt1/2/3, phospho‐Akt1/2/3; T, tumor. NI‐WB (A‐D) was performed on biological replicates (n = 32).

Fig. 3

KRAS mutation‐dependent upregulation of SOX9 in CRC tumor compared to matched mucosa. A representative NI‐WB of SOX9 and ACTB for 7 pairs of CRC tumor and matched mucosa (A). Box and whiskers plot of SOX9/ACTB I.I K counts ratio of mucosa and tumor pairs from NI‐WB (n = 31) (B). Box and whiskers plot of NI‐WB SOX9/ACTB I.I K counts ratio of mucosa and tumor tissues grouped by tumor KRAS mutation status (C), of KRAS WT and pooled mutant tumor (D), of tumor grouped by Duke’s stage (A/B early stage; C/D advanced stage) phospho‐ERK (E) or tumor site (right‐colon; left‐colon) (F). Linear regression of NI‐WB tumor SOX9/ACTB with phospho‐ERK1/total‐ERK1 or phospho‐ERK2/total‐ERK2 I.I K counts ratio (G). Representative confocal images of IHC of SOX9 (red) with nuclear DAPI (blue) counterstain of CRC‐matched mucosa and tumor cryosections for each KRAS mutation status, scale bar = 50 µm in top left image is applicable to all images (H). *one‐tailed t‐test P < 0.05; **two‐tailed t‐test P < 0.05; ****two‐tailed t‐test P < 0.0001. i, paired t‐test; ii, unpaired t‐test with Welch’s correction. M, mucosa; mut, mutant; T, tumor. Both NI‐WB (A–D) and IHC (H) were performed on biological replicates (n = 31 and n = 8, respectively).