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A small molecule targeting NMHC IIA phosphorylation exerts promising anti-tumor effects on colorectal cancer

ABSTRACT: Colorectal cancer (CRC) is one of the most lethal cancers when it progresses to the advanced/metastatic stage. Treatment options for refractory metastatic colorectal cancer (mCRC) are limited. Therefore, there is an urgent need to develop effective treatment methods for metastatic colorectal cancer. In this study, we screened a library of small molecules for inhibitors of CRC recurrence using CRC cell lines and patient-derived organoids (PDOs) from metastatic, heavily pretreated CRC. The in vivo efficacy of LS-1-2 was evaluated in the HCT116 and the resistant HCT8 cell lines, patient-derived xenografts(PDXs) including refractory mCRC, and a liver metastasis mouse model. Biotin pull-down and liquid chromatography tandem-mass spectrometry (LC-MS/MS) were performed to identify proteins that interact with LS-1-2. The genome-wide gene expression and quantitative phosphoproteomic analyses were performed to determine the signaling pathway involved in LS-1-2. Molecular docking, molecular dynamics analysis and cellular thermal shift assays were used to predict and validate the binding sites of LS-1-2 on non-muscle Myosin IIA (NMHC IIA). Our results showed that LS-1-2 exhibited broad antiproliferative effects on a series of CRC cell lines and PDOs. The in vivo anti-tumor efficacy of LS-1-2 was demonstrated across cell line- and patient-derived xenografts and in the liver metastatic CRC model. NMHC IIA was identified as a direct target of LS-1-2, and LS-1-2 competitively inhibited the phosphorylation of NMHC IIA at S1943 and S1714 by CK2. NMHC IIA phosphorylation promoted CRC cell proliferation and invasion, which was reduced by LS-1-2. NMHC IIA phosphorylation-mediated YAP activity induced activation of AKT and inactivation of FOXO3a and was suppressed by LS-1-2. In conclusion, our findings suggest that NMHC IIA phosphorylation can be used as a potential molecular target in CRC metastasis, and that targeting NMHC IIA phosphorylation by LS-1-2 may be a promising strategy for the treatment and prevention of CRC metastasis.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Dengbo Ji

LAB HEAD: Dengbo Ji

PROVIDER: PXD044543 | Pride | 2023-09-02

REPOSITORIES: Pride

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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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A small molecule targeting NMHC IIA phosphorylation exerts promising anti-tumor effects on colorectal cancer

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