Dual blockade of EGFR and ERK1/2 phosphorylation potentiates growth inhibition of breast cancer cells

Lev, Dina; Kim, L. S.; Melnikova, Vladislava O.; Ruiz, Maribelís; Ananthaswamy, Honnavara N.; Price, Janet E.

doi:10.1038/sj.bjc.6602051

Cited by 55 publications

(35 citation statements)

References 55 publications

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“…4 -6). These cell lines have been shown to overexpress the EGF receptor (52). Moreover, we show for the first time that Sam68 is tyrosine phosphorylated downstream of the EGF receptor with slower kinetics than the EGF receptor implying that there is an intermediate protein.…”

Section: Discussionmentioning

confidence: 67%

Tyrosine Phosphorylation of Sam68 by Breast Tumor Kinase Regulates Intranuclear Localization and Cell Cycle Progression

Lukong

Larocque

Tyner

et al. 2005

Journal of Biological Chemistry

122

147

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The breast tumor kinase (BRK) is a growth promoting non-receptor tyrosine kinase overexpressed in the majority of human breast tumors. BRK is known to potentiate the epidermal growth factor (EGF) response in these cells. Although BRK is known to phosphorylate the RNA-binding protein Sam68, the specific tyrosines phosphorylated and the exact role of this phosphorylation remains unknown. Herein, we have generated Sam68 phospho-specific antibodies against C-terminal phosphorylated tyrosine residues within the Sam68 nuclear localization signal. We show that BRK phosphorylates Sam68 on all three tyrosines in the nuclear localization signal. By indirect immunofluorescence we observed that BRK and EGF treatment not only phosphorylates Sam68 but also induces its relocalization. Tyrosine 440 was identified as a principal modulator of Sam68 localization and this site was phosphorylated in response to EGF treatment in human breast tumor cell lines. Moreover, this phosphorylation event was inhibited by BRK small interfering RNA treatment, consistent with Sam68 being a physiological substrate of BRK downstream of the EGF receptor in breast cancer cells. Finally, we observed that Sam68 suppressed BRK-induced cell proliferation, suggesting that Sam68 does indeed contain anti-proliferative properties that may be neutralized in breast cancer cells by phosphorylation.Sam68, initially identified as a c-Src-associated substrate during mitosis of 68 kDa (1, 2), has since been reported to be a substrate of other Src family tyrosine kinases as well as BReast tumor kinase (BRK) 5 (3) and ZAP70 (4). Sam68 is a prototype of STAR proteins, so named for their implication in signal transduction and activation of RNA metabolism (5). STAR proteins contain the GSG (GRP33, Sam68, and GLD-1) or STAR domain, an evolutionarily conserved protein module initially identified by aligning the first three members of this family (6). Although the precise role of Sam68 is unknown, its multifunctionality is revealed by the presence of polyproline sequences, an RNA-binding K homology module shared by all GSG/STAR proteins and multiple potential tyrosine phosphorylation sites (5). The proline-rich sequences mediate interaction with SH3 domains of signaling molecules (7) and the WW domain containing proteins (8). The K homology domain of Sam68 has been shown to bind homopolymeric RNA poly(U), poly(A) (2, 9, 10), and synthetic RNA sequences with a core UAAA (11). However, its in vivo RNA targets have just begun to be identified (12).Several reports have indicated that phosphorylation regulates key cellular roles of Sam68. Both tyrosine phosphorylation and SH3 binding severely hamper the RNA binding capability of Sam68 (13,14). Sam68 was found to interact and colocalize with the splicing-associated factor YT512-B, to synergize with the human immunodeficiency virus Rev protein, enhancing export of unspliced viral RNA, and to increase protein expression from RNA containing the constitutive transport element of some retroviruses (3,(15)(16)(17). On the other han...

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Section: Discussionmentioning

confidence: 67%

Tyrosine Phosphorylation of Sam68 by Breast Tumor Kinase Regulates Intranuclear Localization and Cell Cycle Progression

Lukong

Larocque

Tyner

et al. 2005

Journal of Biological Chemistry

122

147

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“…Moreover, the results of this study showed that rare and potentially "druggable" oncogenic mutations may exist in many common tumor types regarded as WT against well-known oncogenes (20). Thus, there is a clear need for more rational therapeutic strategies of genetically complex tumor cells, and several studies have already provided initial rational combination therapies (22)(23)(24). In this study, we characterize AZD6244 (ARRY-142886), a novel MAP/ERK kinase (MEK) inhibitor that exerts antiproliferative effects on human gastric cancer cell lines that are resistant to gefitinib.…”

Section: Introductionmentioning

confidence: 84%

“…EGFR (exons [18][19][20][21][22][23][24], KRAS (exons 1-3), and BRAF (exon 15) were then sequenced using the previously described primers and methods (10,25,26). All sequencing reactions were conducted in the forward and reverse directions.…”

Section: Methodsmentioning

confidence: 99%

Combination of EGFR and MEK1/2 inhibitor shows synergistic effects by suppressing EGFR/HER3-dependent AKT activation in human gastric cancer cells

Yoon

Kim

Han

et al. 2009

Molecular Cancer Therapeutics

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EGFR tyrosine kinase inhibitors have shown promising efficacy in the treatment of tumors with EGFR mutations and amplifications. However, tyrosine kinase inhibitors have also proven ineffective against most tumors with EGFR wild-type (WT) alleles. Although some genetic changes, including the KRAS mutation, have been shown to confer resistance to tyrosine kinase inhibitors, novel strategies for the treatment of cancer patients with tumors harboring EGFR WT alleles have yet to be thoroughly delineated. The principal objective of this study was to improve our current understanding of drug interactions between EGFR and MAP/ERK kinase (MEK) inhibitors in an effort to gain insight into a novel therapeutic strategy against EGFR WT tumors. Using a panel of human EGFR WT gastric cancer cell lines, we showed that gastric cancer cells harboring the KRAS mutation were selectively sensitive to MEK inhibition as compared with those cells harboring KRAS and PI3K mutations and KRAS WT alleles. However, all cell lines were found to be resistant to EGFR inhibition. The results from Western blots and phosphoprotein arrays showed that, in MEK inhibitor resistant cell lines, AKT was activated through the EGFR/HER3/PI3K pathway following AZD6244 (ARRY-142886) treatment. Blockade of this feedback mechanism through the targeting of MEK and EGFR resulted in detectable synergistic effects in some cell lines in vitro and in vivo. Our results provide the basis for a rational combination strategy against human EGFR WT gastric cancers, predicated on the understanding of cross-talk between the MEK and EGFR pathways. [Mol Cancer Ther 2009;8(9):2526-36]

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“…Proliferation of the MDA-MB-231 line is dependent on PI3K/Akt pathways (15), whereas the extracellular signalregulated kinase inhibitor U0126 exhibits little or no inhibition of this line (37), suggesting that the MDA-MB-231 line is a candidate to study for ritonavir-associated Akt inhibition in a xenograft model. To test the effectiveness of ritonavir treatment on MDA-MB-231 xenografts, nude mice were implanted with 1 Â 10 6 tumor cells and the tumors were established for 3 weeks before treatment with ritonavir.…”

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confidence: 99%

Effects of HIV Protease Inhibitor Ritonavir on Akt-Regulated Cell Proliferation in Breast Cancer

Srirangam

Mitra

Wang

et al. 2006

Clinical Cancer Research

103

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Purpose: These studies were designed to determine whether ritonavir inhibits breast cancer in vitro and in vivo and, if so, how. Experimental Design: Ritonavir effects on breast cancer cell growth were studied in the estrogen receptor (ER)^positive lines MCF7 and T47D and in the ER-negative lines MDA-MB-436 and MDA-MB-231. Effects of ritonavir on Rb-regulated and Akt-mediated cell proliferation were studied. Ritonavir was tested for inhibition of a mammary carcinoma xenograft. Results: ER-positive estradiol-dependent lines (IC 50 , 12-24 Amol/L) and ER-negative (IC 50 , 45 Amol/L) lines exhibit ritonavir sensitivity. Ritonavir depletes ER-a levels notably in ER-positive lines. Ritonavir causes G 1 arrest, depletes cyclin-dependent kinases 2, 4, and 6 and cyclin D 1 but not cyclin E, and depletes phosphorylated Rb and Ser 473 Akt. Ritonavir induces apoptosis independent of G 1 arrest, inhibiting growth of cells that have passed the G 1 checkpoint. Myristoyl-Akt, but not activated K-Ras, rescues ritonavir inhibition. Ritonavir inhibited a MDA-MB-231 xenograft and intratumoral Akt activity at a clinically attainable serum C max of 22 F 8 Amol/L. Because heat shock protein 90 (Hsp90) substrates are depleted by ritonavir, ritonavir effects on Hsp90 were tested. Ritonavir binds Hsp90 (K D , 7.8 Amol/L) and partially inhibits its chaperone function. Ritonavir blocks association of Hsp90 with Akt and, with sustained exposure, notably depletes Hsp90. Stably expressed Hsp90a short hairpin RNA also depletes Hsp90, inhibiting proliferation and sensitizing breast cancer cells to low ritonavir concentrations. Conclusions: Ritonavir inhibits breast cancer growth in part by inhibiting Hsp90 substrates, including Akt. Ritonavir may be of interest for breast cancer therapeutics and its efficacy may be increased by sustained exposure or Hsp90 RNA interference.

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Dual blockade of EGFR and ERK1/2 phosphorylation potentiates growth inhibition of breast cancer cells

Cited by 55 publications

References 55 publications

Tyrosine Phosphorylation of Sam68 by Breast Tumor Kinase Regulates Intranuclear Localization and Cell Cycle Progression

Tyrosine Phosphorylation of Sam68 by Breast Tumor Kinase Regulates Intranuclear Localization and Cell Cycle Progression

Combination of EGFR and MEK1/2 inhibitor shows synergistic effects by suppressing EGFR/HER3-dependent AKT activation in human gastric cancer cells

Effects of HIV Protease Inhibitor Ritonavir on Akt-Regulated Cell Proliferation in Breast Cancer

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