Functional identification of genes causing estrogen independence of human breast cancer cells

Agthoven, Ton van; Veldscholte, Jos; Smid, Marcel; Agthoven, Thecla L. A. van; Vreede, Lilian; Broertjes, Marieke; Vries, Ingrid de; Jong, Daniëlle de; Sarwari, Roya; Dorssers, Lambert C. J.

doi:10.1007/s10549-008-9969-5

Cited by 44 publications

(39 citation statements)

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“…In the experiments using the ZR-75-1 cell line as a target, all cell clones recovered from the different cultures were found to contain an integrated retrovirus, in agreement with the documented selectivity of this model (Dorssers et al 1993, Van Agthoven et al 2009b. From a total of 264 cell clones isolated from these selective cultures, 132 different inserted genes were identified (Table 1 and Supplementary Table S2, see section on supplementary data given at the end of this article).…”

Section: Functional Screen Identifies Novel Bcar Genessupporting

confidence: 66%

“…Our transfection experiments have shown that many BCAR genes provide resistance to tamoxifen as well as pure anti-estrogens (Van Agthoven et al 2009b). However, in a screen based on colony formation using different culture conditions, the efficiency of individual genes may vary substantially.…”

Section: Discussionmentioning

confidence: 95%

“…The coding regions of HRAS (corresponding to nt 11 to 1029 of NM_005343.2), RAF1 (nt 883 to 2489 of NM_002880), PDGFRA (NM_006206), and PDGFRB (NM_002609.1) were cloned into the LZRS-IRES-Neo expression vector and introduced into ZR-75-1 cells as described (Meijer et al 2006, Van Agthoven et al 2009b. Two independently generated pools of cells were assayed for anti-estrogen resistance in 96-well plates using the WST-1 reagent (Roche Diagnostics) according to the recommendation of the supplier.…”

Section: Expression Constructs and Proliferation Assaysmentioning

confidence: 99%

“…This is to ascertain that abundantly expressed genes, which are recovered from cell clones as passengers and are not responsible for the resistance, are not designated as BCAR genes. These criteria also preclude the possibility that insertion of a cDNAcontaining virus leading to an insertion mutagenesis event, which may occur at a frequency of w1 per 10 million infected cells (Van Agthoven et al 2009b), is mistaken for a BCAR gene. Based on these criteria, our studies identified a total of 15 BCAR genes (ABCB1, BCAR4, CSFR1, EEF1A1, EGFR, EIF1, FBXL10, FGF17, HRAS, NRG1, PDGFRA, PDGFRB, RAD21, RAF1, and RPL18A) individually responsible for anti-estrogen-resistant cell proliferation (Table 2).…”

Section: Functional Screen Identifies Novel Bcar Genesmentioning

confidence: 99%

“…The integration of virus can affect an individual gene and thereby altering the cell phenotype (Dorssers et al 1993). Through this approach, seven breast cancer anti-estrogen resistance (BCAR) genes responsible for the transition of an estrogen-dependent, tamoxifen-sensitive human breast cancer cell line into a tamoxifen-resistant phenotype have been elucidated (Van Agthoven et al 1998, 2009b, Brinkman et al 2000. In view of the complexity and the workload of this approach, we have recently tested retroviral transduction of cDNA expression libraries (Brummelkamp & Bernards 2003) as an alternative strategy.…”

Section: Introductionmentioning

confidence: 99%

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Selective recruitment of breast cancer anti-estrogen resistance genes and relevance for breast cancer progression and tamoxifen therapy response

Agthoven¹,

Sieuwerts²,

Meijer³

et al. 2010

Endocrine-Related Cancer

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Although endocrine treatment of breast cancer is effective and common practice, in advanced disease the development of resistance is nearly inevitable. To get more insight into individual genes that account for resistance against hormonal agents, we have executed functional genetic screens and subsequently evaluated the clinical relevance of several identified genes with respect to tumor aggressiveness and tamoxifen resistance in estrogen receptor-positive patients. Estrogen-dependent human breast cancer cells were transduced with different retroviral cDNA expression libraries and subjected to selective cultures with various anti-estrogens. From a total of 264 resistant cell clones, 132 different genes were recovered by PCR. By applying stringent selection criteria, we identified 15 breast cancer anti-estrogen resistance (BCAR) genes individually yielding resistance. BCAR genes were recovered with differential frequencies for the diverse culture conditions and anti-estrogen drugs. Analysis of the relation of BCAR genes (EIF1, FBXL10, HRAS, NRG1, PDGFRA, PDGFRB, RAD21, and RAF1) with tamoxifen treatment in patients with advanced disease showed significant association with clinical benefit and progression-free survival for EIF1 and PDGFRA mRNA levels. Furthermore, PDGFRA and HRAS mRNA levels were significantly associated with tumor aggressiveness in lymph node-negative patients who had not received adjuvant systemic therapy. In conclusion, our functional genetic screens showed that BCAR genes differ in their ability to confer resistance towards distinct antiestrogens. Based on the clinical relevance of several BCAR genes, further studies are warranted to characterize the underlying mechanisms, which may ultimately lead to the development of novel treatments and more individualized management of breast cancer patients.

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Section: Functional Screen Identifies Novel Bcar Genessupporting

confidence: 66%

Section: Discussionmentioning

confidence: 95%

Section: Expression Constructs and Proliferation Assaysmentioning

confidence: 99%

Section: Functional Screen Identifies Novel Bcar Genesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Selective recruitment of breast cancer anti-estrogen resistance genes and relevance for breast cancer progression and tamoxifen therapy response

Agthoven¹,

Sieuwerts²,

Meijer³

et al. 2010

Endocrine-Related Cancer

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Protein pathway activation mapping reveals molecular networks associated with antiestrogen resistance in breast cancer cell lines

Agthoven

Godinho

Wulfkuhle

et al. 2012

Intl Journal of Cancer

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Previously, we have identified a panel of breast cancer antiestrogen resistance (BCAR) genes. Several of these genes have clinical relevance because mRNA or protein levels associate with tamoxifen resistance or tumor aggressiveness. We postulated that changes in activation status of protein signaling networks induced by BCAR genes may provide better insight into the mechanisms underlying antiestrogen resistance. Key signal transduction pathways were analyzed for changes in activation or expression using reverse-phase protein microarrays probed with 78 antibodies against signaling proteins with known roles in tumorigenesis. We used ZR-75-1-derived cell lines transduced with AKT1, AKT2, BCAR1, BCAR3, BCAR4, EGFR, GRB7, HRAS, HRAS v12 or HEF1 and MCF7-derived cell lines transduced with BCAR3, BCAR4 or EGFR. In the antiestrogenresistant cell lines, we observed increased phosphorylation of several pathways involved in cell proliferation and survival. All tamoxifen-resistant cell lines contained high levels of phosphorylated AKT and its biochemically linked substrates Forkhead box O1/3. The activation of ERBB2, ERBB3 and the downstream modulators focal adhesion kinase and SHC were activated in cells with overexpression of BCAR4. Remarkable differences were observed for the levels of activated AMPK alpha1, cyclins, STAT5, STAT6, ERK1/2 and BCL2. The comparison of the cell signaling networks in estrogen-dependent and -independent cell lines revealed biochemically linked kinase-substrate markers that comprised systemically activated signaling pathways involved in tamoxifen resistance. Our results show that this model provides insights into the molecular and cellular mechanisms of breast cancer progression and antiestrogen resistance. This knowledge may help the development of novel targeted treatments.Breast cancers are initially dependent on estrogens for growth and progression. Antiestrogens prevent estrogens from binding the estrogen receptor (ER) alpha and thereby inhibit cell proliferation. ERa is expressed in about two-thirds of primary breast cancers. For over 30 years, the estrogen antagonist tamoxifen has been the preferred endocrine treatment for patients with ERa-positive breast cancer in premenopausal and postmenopausal women. 1 Tamoxifen is effective in the treatment of metastatic breast cancer, ductal carcinoma in situ, as adjuvant therapy, and more recently in chemoprevention of the malignancy. 2,3 In patients with breast cancer with recurrent disease, tamoxifen induces an objective response in approximately more than half of the patients with primary cancers expressing the ER 4 and references herein. However, the treatment is palliative, and progression of the tumor cells to a hormone-independent phenotype is inevitable. Several mechanisms have been identified underlying primary endocrine resistance of breast cancer and the progression to acquired resistance. The most favored explanations for mechanisms causing tamoxifen resistance are based on pharmacology of the drug; alterations in the interactions bet...

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Expression of a phosphorylated p130^Cas substrate domain attenuates the phosphatidylinositol 3‐kinase/Akt survival pathway in tamoxifen resistant breast cancer cells

Soni¹,

Lin²,

August³

et al. 2009

J of Cellular Biochemistry

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Elevated expression of p130Cas/BCAR1 (breast cancer anti estrogen resistance 1) in human breast tumors is a marker of poor prognosis and poor overall survival. Specifically, p130Cas signaling has been associated with antiestrogen resistance, for which the mechanism is currently unknown. TAM-R cells, which were established by long-term exposure of estrogen (E2)-dependent MCF-7 cells to tamoxifen, displayed elevated levels of total and activated p130Cas. Here we have investigated the effects of p130Cas inhibition on growth factor signaling in tamoxifen resistance. To inhibit p130Cas, a phosphorylated substrate domain of p130Cas, that acts as a dominant-negative (DN) p130Cas molecule by blocking signal transduction downstream of the p130Cas substrate domain, as well as knockdown by siRNA was employed. Interference with p130Cas signaling/expression induced morphological changes, which were consistent with a more epithelial-like phenotype. The phenotypic reversion was accompanied by reduced migration, attenuation of the ERK and phosphatidylinositol 3-kinase/Akt pathways, and induction of apoptosis. Apoptosis was accompanied by downregulation of the expression of the anti-apoptotic protein Bcl-2. Importantly, these changes re-sensitized TAM-R cells to tamoxifen treatment by inducing cell death. Therefore, our findings suggest that targeting the product of the BCAR1 gene by a peptide which mimics the phosphorylated substrate domain may provide a new molecular avenue for treatment of antiestrogen resistant breast cancers.

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Functional identification of genes causing estrogen independence of human breast cancer cells

Cited by 44 publications

References 47 publications

Selective recruitment of breast cancer anti-estrogen resistance genes and relevance for breast cancer progression and tamoxifen therapy response

Selective recruitment of breast cancer anti-estrogen resistance genes and relevance for breast cancer progression and tamoxifen therapy response

Protein pathway activation mapping reveals molecular networks associated with antiestrogen resistance in breast cancer cell lines

Expression of a phosphorylated p130^Cas substrate domain attenuates the phosphatidylinositol 3‐kinase/Akt survival pathway in tamoxifen resistant breast cancer cells

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Functional identification of genes causing estrogen independence of human breast cancer cells

Cited by 44 publications

References 47 publications

Selective recruitment of breast cancer anti-estrogen resistance genes and relevance for breast cancer progression and tamoxifen therapy response

Selective recruitment of breast cancer anti-estrogen resistance genes and relevance for breast cancer progression and tamoxifen therapy response

Protein pathway activation mapping reveals molecular networks associated with antiestrogen resistance in breast cancer cell lines

Expression of a phosphorylated p130Cas substrate domain attenuates the phosphatidylinositol 3‐kinase/Akt survival pathway in tamoxifen resistant breast cancer cells

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Expression of a phosphorylated p130^Cas substrate domain attenuates the phosphatidylinositol 3‐kinase/Akt survival pathway in tamoxifen resistant breast cancer cells