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Overexpression of KRAS-Q25A mutant with enhanced GTPase activity inhibits the progression of KRAS-G12D colorectal cancer

ABSTRACT: The high mutation rate of KRAS in cancers leads to consistent activation of downstream signaling and eventually promoting malignancy. While restoring GTP hydrolysis in KRAS-mutant tumors represents a promising therapeutic strategy, its mechanistic basis remains unclear. Our previous finding that mutation of the KRAS P4 pocket exhibited elevated intrinsic GTPase activity of KRAS, and therefore presented a promising new strategy to intervene KRAS mutated cancer. This exciting potential has now been verified in the current work. We explored how P4 mutants impact the progression of colorectal cancer, and found KRAS-Q25A, a P4 mutant with the most significant elevated GTPase activity, can inhibit the progression of CT26 cell, a commonly used colorectal cell line with KRAS-G12D mutation. Moreover, this inhibitory effect of KRAS-Q25A is depended on its GTP hydrolysis process. To assess the impact of KRAS-Q25A overexpression on the overall cellular state, we performed RNA sequencing on CT26 cells overexpressing either KRAS-WT or KRAS-Q25A.

ORGANISM(S): Mus musculus

PROVIDER: GSE332549 | GEO | 2026/05/21

REPOSITORIES: GEO

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Project description:KRAS mutation is a negative predictive factor for treatment with anti-epidermal growth factor receptor (EGFR) antibodies in metastatic colorectal cancer (mCRC). Novel predictive markers are required to further improve the selection of patients for this treatment. Here, we assessed the influence of modification of KRAS by gene copy number aberration (CNA) and microRNAs (miRNAs) in correlation to clinical outcome in mCRC patients treated with cetuximab in combination with chemotherapy and bevacizumab. Formalin-fixed paraffin-embedded primary tumour tissue was used from 34 mCRC patients in a phase III trial, who were selected based upon their good (n=17) or poor (n=17) progression-free survival (PFS) upon treatment with cetuximab in combination with capecitabine, oxaliplatin, and bevacizumab. Gene copy number at the KRAS locus was assessed using high resolution genome-wide array CGH and the expression levels of 17 miRNAs targeting KRAS were determined by real-time PCR. Good response was associated with 12p12.1 copy number loss, even in patients with a KRAS mutation, while copy number gain in wild-type KRAS patients was correlated with a poor response. In KRAS mutated tumours increased miR-200b and decreased miR-143 expression were associated with a good response. In wild-type KRAS patients, miRNA expression did not predict response in a multivariate model. Thus, assessment of KRAS CNA and miRNAs targeting KRAS might further optimize the selection of patients eligible for anti-EGFR therapy. Copy number detection was performed using NimbleScan and Nexus software Formalin-fixed paraffin-embedded primary tumour tissue was used from 34 metastisized colorectal cancer patients in a phase III trial (CKTO 2005-02; ClinTrials.gov NCT00208546) of the Dutch Colorectal Cancer Group (DCCG), who were selected based upon their good (n=17) or poor (n=17) progression-free survival (PFS) upon treatment with cetuximab in combination with capecitabine, oxaliplatin, and bevacizumab.

Project description:Colorectal cancer (CRC) is still one of the most common causes of cancer-related death worldwide (World Health Organization, Fact sheet NM-BM-0297, October 2011). Though, therapeutic options improved over the past years, the prognosis of metastatic colorectal cancer (mCRC) remains poor with a median survival of 18-21 months. Monoclonal antibodies targeting the epidermal growth factor receptor (EGFR) are used for the palliative treatment of metastatic colorectal cancer (mCRC) carrying a KRAS-wildtype. Among the tumors with a KRAS-wildtype, only 35% respond, and alterations of other signaling molecules and pathways modulate therapeutic response. In this study we analyzed the effect of KRAS-mutations on intracellular signaling cascades to find new therapeutic approaches for patients with mCRC. Reverse-phase protein array and gene array technology was applied to sixteen adenocarcinomas of the sigmoid colon carrying either wildtype (n=8) or mutant (n=8) KRAS. Differential expression was validated on 49 sigmoid cancers by RT-PCR, Western blot and immunohistochemistry. In a screening for members of signaling pathways known to have a high impact on tumor development in colon cancer, we found 14 proteins downregulated in tumors with mutated KRAS. Subsequently, we compared the data of the protein arrays with the results of gene expression arrays performed with the same samples. We confirmed the differential expression of eight genes as a function of the KRAS genotype of the tumor. To further validate the differential expression of these candidates, we performed quantitative expression analysis and immunohistochemical staining of an independent validation series comprising 49 CRC patient samples. It was clearly demonstrated that the expression of EGFR is decreased in colorectal carcinomas with a mutant KRAS genotype. mRNA was obtained from 16 patients with colorectal cancer (8 with and 8 without KRAS-mutation). mRNA was extracted from neoplastic and non-neoplastic colon mucosa and analyzed separately by Affymetrix Human Gene 1.1 ST GeneChips finally generating 32 separate data sets.

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Overexpression of KRAS-Q25A mutant with enhanced GTPase activity inhibits the progression of KRAS-G12D colorectal cancer

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