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Functional interactions between Choline kinase ?, epidermal growth factor receptor and c-Src in breast cancer cell proliferation.

ABSTRACT: Epidermal growth factor receptor (EGFR) family members and c-Src are co-overexpressed in many cancers. The synergistic effect of EGFR and c-Src has been shown in the tumorigenesis of breast and other cancers. Reported mechanisms of synergy include transcriptional regulation by STAT5b and the regulation of cellular ATP production by mitochondrial protein COX II. Here, we report a new mechanism of EGFR-c-Src synergy through choline kinase ? (CHKA). The first enzyme of the phosphatidyl choline production pathway, CHKA, is overexpressed in many cancers, and the product of the enzyme, phosphocholine, is also increased in tumor cells. In this report, we find that CHKA forms a complex with EGFR in a c-Src-dependent manner. Endogenous CHKA and EGFR co-immunoprecipitated from a variety of breast cancer cell lines and immortalized mammary epithelial cells. CHKA interacted with the EGFR kinase domain upon c-Src co-overexpression and was phosphorylated in a c-Src-dependent manner on Y197 and Y333. Overexpression of EGFR and c-Src increased total cellular activity and protein levels of CHKA. Mutation of CHKA Y197 and Y333 reduced complex formation, EGFR-dependent activation of CHKA enzyme activity and epidermal growth factor (EGF)-dependent DNA synthesis. Furthermore, small interfering RNA-mediated knockdown of CHKA in MCF-7 and MCF-10A cells reduced EGF-dependent cell proliferation. Together, these results strongly implicate a new c-Src-dependent link between CHKA and EGFR, which contributes to the regulation of cell proliferation and tumorigenesis.

SUBMITTER: Miyake T

PROVIDER: S-EPMC3213328 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Functional interactions between Choline kinase α, epidermal growth factor receptor and c-Src in breast cancer cell proliferation.

Miyake T T Parsons S J SJ

Oncogene 20110808 11

Epidermal growth factor receptor (EGFR) family members and c-Src are co-overexpressed in many cancers. The synergistic effect of EGFR and c-Src has been shown in the tumorigenesis of breast and other cancers. Reported mechanisms of synergy include transcriptional regulation by STAT5b and the regulation of cellular ATP production by mitochondrial protein COX II. Here, we report a new mechanism of EGFR-c-Src synergy through choline kinase α (CHKA). The first enzyme of the phosphatidyl choline prod ...[more]

PMID: 21822308

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Project description:BACKGROUND & AIMS:Choline kinase ? (CHKA) catalyzes conversion of choline to phosphocholine and can contribute to carcinogenesis. Little is known about the role of CHKA in the pathogenesis of hepatocellular carcinoma (HCC). METHODS:We performed whole-exome and transcriptome sequence analyses of 9 paired HCC and non-tumor-adjacent tissues. We performed tissue chip analyses of 120 primary HCC and non-tumor-adjacent tissues from patients who received surgery in Shanghai, China from January 2006 through December 2009; 48 sets of specimens (HCC and non-tumor-adjacent tissues) were also analyzed. CHKA gene copy number was quantified and findings were validated by quantitative reverse transcription polymerase chain reaction analysis. CHKA messenger RNA and protein levels were determined by polymerase chain reaction, immunohistochemical, and immunoblot analyses. CHKA was examined in 2 hepatocyte cell lines and 7 HCC-derived cell lines, and knocked down with small interfering RNAs in 3 HCC cell lines. Cells were analyzed in proliferation, wound healing, migration, and invasion assays. Cells were injected into tail veins of mice and tumor growth and metastasis were quantified. Immunoprecipitation and immunofluorescence assays were conducted to determine interactions between CHKA and the epidermal growth factor receptor (EGFR) and the mechanistic target of rapamycin complex 2. RESULTS:Levels of CHKA messenger RNA were frequently increased in HCC tissues compared with nontumor tissues; increased expression was associated with amplification at the CHKA loci. Tumors that expressed high levels of CHKA had more aggressive phenotypes, and patients with these tumors had shorter survival times after surgery compared to patients whose tumors expressed low levels of CHKA. HCC cell lines that stably overexpressed CHKA had higher levels of migration and invasion than control HCC cells, and formed larger xenograft tumors with more metastases in mice compared to HCC cells that did not overexpress CHKA. CHKA was required for physical interaction between EGFR and mechanistic target of rapamycin complex 2. This complex was required for HCC cells to form metastatic xenograft tumors in mice and to become resistant to EGFR inhibitors. CONCLUSIONS:We found levels of CHKA to be increased in human HCCs compared to nontumor tissues, and increased expression to be associated with tumor aggressiveness and reduced survival times of patients. Overexpression of CHKA in HCC cell lines increased their invasiveness, resistance to EGFR inhibitors, and ability to form metastatic tumors in mice by promoting interaction of EGFR with mechanistic target of rapamycin complex 2.

Project description:BackgroundNew molecular targets are needed for women with triple-negative breast cancer (TNBC). This pre-clinical study investigated the combination of the EGFR inhibitor gefitinib with the sphingosine kinase (SphK) inhibitor FTY720 (Fingolimod), aiming to block tumorigenic signaling downstream of IGFBP-3, which is abundantly expressed in basal-like TNBC.MethodsIn studies of breast cancer cell growth in culture, proliferation was monitored by IncuCyte live-cell imaging, and protein abundance was determined by western blotting. In vivo studies of mammary tumor growth used two models: orthotopic xenograft tumors derived from three basal-like TNBC cell lines, grown in immune-deficient mice, and syngeneic murine 4T1 tumors grown in immune-competent mice. Protein abundance in tumor tissue was assessed by immunohistochemistry.ResultsQuantitated by live-cell imaging, the inhibitor combination showed synergistic cytostatic activity in basal-like cell lines across several TNBC molecular subtypes, the synergy being decreased by IGFBP-3 downregulation. Suppression of the tumorigenic mediator CD44 by gefitinib was potentiated by FTY720, consistent with CD44 involvement in the targeted pathway. In MDA-MB-468 and HCC1806 orthotopic TNBC xenograft tumors in nude mice, the drug combination inhibited tumor growth and prolonged mouse survival, although this effect was not significant for the gefitinib-resistant cell line HCC70. Combination treatment of murine 4T1 TNBC tumors in syngeneic BALB/c mice was more effective in immune-competent than immune-deficient (nude) mice, and a relative loss of tumor CD3 (T-cell) immunoreactivity caused by FTY720 treatment alone was alleviated by the drug combination, suggesting that, even at an FTY720 dose causing relative lymphopenia, the combination is still effective in an immune-competent setting. Immunohistochemistry of xenograft tumors showed significant enhancement of caspase-3 cleavage and suppression of Ki67 and phospho-EGFR by the drug combination, but SphK1 downregulation occurred only in MDA-MB-468 tumors, so is unlikely to be integral to treatment efficacy.ConclusionsOur data indicate that targeting IGFBP-3-dependent signaling pathways through gefitinib-FTY720 co-therapy may be effective in many basal-like breast cancers, and suggest tissue IGFBP-3 and CD44 measurement as potential biomarkers of treatment efficacy.

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Functional interactions between Choline kinase ?, epidermal growth factor receptor and c-Src in breast cancer cell proliferation.

Publications

Functional interactions between Choline kinase α, epidermal growth factor receptor and c-Src in breast cancer cell proliferation.

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