Constitutive Raf-1 kinase activity in breast cancer cells induces both estrogen-independent growth and apoptosis

El-Ashry, Dorraya; Miller, David L.; Kharbanda, Samir; Lippman, Marc E.; Kern, Florian

doi:10.1038/sj.onc.1201198

Cited by 96 publications

(64 citation statements)

References 27 publications

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“…All cell lines derived from MCF-7 cells via transfection or selection grow in an estrogen-independent manner (Clarke et al, 1994;El-Ashry et al, 1997;Kern et al, 1994). However, all but one (Raf14c) continue to express ER protein.…”

Section: Discussionmentioning

confidence: 99%

“…Eight additional cell lines derived from the ERpositive MCF-7 line were also examined ( Figure 5). Raf14c (overexpressing a constitutively active form of Raf) and its vector control, HCopoolc, as well as MKL4 and a18 cells (which overexpress FGF4 and FGF1 respectively) and their vector control, MCN4 were provided by Dr Dorraya ElAshry and Dr Francis Kern of the Lombardi Cancer Center and Southern Research Institute, respectively (El-Ashry et al, 1997;Kern et al, 1994). These transfected lines have acquired estrogen independent growth as a result of their speci®c gene overexpression and are grown continuously in the absence of estrogen.…”

Section: Cell Linesmentioning

confidence: 99%

“…These transfected lines have acquired estrogen independent growth as a result of their speci®c gene overexpression and are grown continuously in the absence of estrogen. The MCN4 and HCopoolc cell lines were selected in vitro for estrogen independent growth over a period of about nine months (El-Ashry et al, 1997;Kern et al, 1994). MCF-7/MIII, MCF-7/LCCI, and MCF-7/LCC2 (Clarke et al, 1994) were a gift from Dr Robert Clarke (Lombardi Cancer Center, Washington DC, USA).…”

Section: Cell Linesmentioning

confidence: 99%

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Expression of DNA methyl-transferase (DMT) and the cell cycle in human breast cancer cells

et al. 1999

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Estrogen receptor (ER)-negative breast cancer cells display extensive methylation of the ER gene CpG island and elevated DNA methyltransferase (DMT) expression compared to ER-positive cells. The present study demonstrates that DMT protein levels tightly correlate with S phase fraction in ER-positive cells, whereas ER-negative cells express DMT throughout the cell cycle. In addition, levels of p21 CIP1 , which disrupts DMT binding to PCNA, are inversely correlated with DMT levels. Therefore increased DMT expression in ER-negative cells is not simply due to elevated S-phase fraction, but rather to more complex changes that allow cells to escape normal cell cycle-dependent controls on DMT expression. Because ER-negative breast tumors often have activated growth factor pathways, the impact of these pathways on DMT expression was examined in ER-positive cells. Stable transfection with ®broblast growth factors (FGFs) 1 and 4 led to increased DMT expression that could not be accounted for by a shift in S phase fraction. Elevated DMT protein expression in FGF-transfectants was accompanied by a signi®cant decrease in p21, again suggesting a reciprocal relationship between these two proteins. However, acquisition of an estrogen-independent phenotype, even in conjunction with elevated DMT levels, was not su cient to promote ER gene silencing via methylation. These results indicate that multiple steps are required for de novo methylation of the ER CpG island.

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Section: Discussionmentioning

confidence: 99%

Section: Cell Linesmentioning

confidence: 99%

Section: Cell Linesmentioning

confidence: 99%

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Expression of DNA methyl-transferase (DMT) and the cell cycle in human breast cancer cells

et al. 1999

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“…Recent evidence has unequivocally shown that the development and progression of endocrine resistant breast cancer involves complex interactions between steroidal and growth factor signalling pathways [4,5]. In particular, increased EGF-R and hyper-activation of its down-stream signalling elements (Ras, MEK-1, ERK 1/2) invariably correlate with hormonal resistance, aggressive clinicopathology, metastatic disease and poor prognosis [6][7][8][9]. Indeed, in MCF-7 breast cancer cell lines displaying acquired resistance to the growth inhibitory effects of either tamoxifen, or the pure anti-oestrogen fulvestrant ('Faslodex'), increased EGFR/c-erbB2/ERK signalling activity is evident.…”

Section: Introductionmentioning

confidence: 99%

Growth of hormone-dependent MCF-7 breast cancer cells is promoted by constitutive caveolin-1 whose expression is lost in an EGF-R-mediated manner during development of tamoxifen resistance

Thomas

Hutcheson

Campbell

et al. 2009

Breast Cancer Res Treat

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Growth of hormone-dependent MCF-7 breast cancer cells is promoted by constitutive caveolin-1 whose expression is lost in an EGF-R-mediated manner during development of tamoxifen resistance. Breast Cancer Research and Treatment, Springer Verlag, 2009, 119 (3) Abstract Caveolin-1 displays both tumour-suppressor and tumour-promoter properties in breast cancer. Using characterised preclinical cell models for the transition of oestrogen-sensitive (WT-MCF-7 cells) to a tamoxifenresistant (TAM-R cells) phenotype we examined the role caveolin-1 in the development of hormone-resistant breast cancer. The WT-MCF-7 cells showed abundant expression of caveolin-1 which potentiated oestrogen-receptor (ERa) signalling and promoted cell growth despite caveolin-1 mediating inhibition of ERK signalling. In TAM-R cells caveolin-1 expression was negligible, repressed by EGF-R/ERK signalling. Pharmacological inhibition of EGFR/ERK in TAM-R cells restored caveolin-1 and also resulted in the emergence of pools of phosphorylated caveolin-1. WT-MCF-7 cells exposed to tamoxifen for upto 12 weeks displayed increased caveolin-1 (peaking by week 2) followed (after week 8) by a marked decrease as the cells progress to develop a stable tamoxifen-resistant phenotype. The targeted down-regulation (siRNA) of caveolin-1 in WT-MCF-7 cells reduced growth but did not affect their sensitivity to tamoxifen, suggesting loss of caveolin-1 alone is not sufficient to confer tamoxifen-resistance. Hyperactivation of EGFR/ERK is a feature of tamoxifen-resistant breast cancer cells, a principal driver of cell growth. Recombinant expression of caveolin-1 in TAM-R cells did not affect EGFR/ERK activity, potentially due to mislocalisation of caveolin-1 through hyperactivation of the mTOR pathway or altered caveolin-1 phosphorylation. This work defines a novel role for caveolin-1 with implications for the clinical course of breast cancer and identifies caveolin-1 as a potential drug target for the treatment of early oestrogen-dependent breast cancers. Further, the loss of caveolin-1 may have benefit as a molecular signature for tamoxifen resistance.

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“…Thus, with each of these cell lines we observed clear morphological alterations to the nucleus (Figure 3a), DNA fragmentation (Figure 3b), staining with FITC-annexin V ( Figure 4a) and PARP cleavage (Figure 4b). Previous studies have implicated Raf proteins in the protection of cells from apoptosis (Cleveland et al, 1994;Wang et al, 1994;Weissinger et al, 1997;Wojnowski et al, 1997) as well as in the induction of PCD (Blagosklonny et al, 1996;El-Ashry et al, 1997). Apart from family member-speci®c di erences among c-Raf/Raf-1, v-Raf and B-Raf, these apparent discrepancies might re¯ect cell type-speci®c functions of, and e ects on, Raf-controlled signal transduction cascades.…”

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confidence: 99%

Abrogation of c-Raf expression induces apoptosis in tumor cells

1998

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Signal transduction pathways involving the c-Raf protein kinase are frequently activated in tumor cells. We have addressed the relevance of this activation by a loss-offunction approach. An anti-sense phosphorothioate oligonucleotide (ODN) speci®cally targeted against craf mRNA (Monia et al., 1996a) was used to block cRaf protein expression in four di erent cell lines derived from lung, cervical, prostate and colon carcinomas. Concomitant with the abrogation of c-Raf expression we observed the occurrence of classical apoptotic markers, including chromatin condensation, inter-nucleosomal DNA cleavage, annexin V binding and cleavage of PARP, which was followed by cell death, a ecting most of the cell population. This induction of apoptosis occurred independent of the p53 status of the cell. These ®ndings demonstrate that c-Raf can protect tumor cells from undergoing programmed cell death, and suggest that the interference with c-Raf expression or function by ODNs or speci®c drugs could represent a powerful means for improving the e cacy of anti-cancer therapy.Keywords: apoptosis; programmed cell death; Raf; antisense; phosphorothiate ODN The occurrence of pro-apoptotic lesions, including the deregulation of Myc or the loss of pRB, is a hallmark of oncogenesis (Evan et al., 1992). The onset of programmed cell death (PCD) is prevented, however, by the concomitant activation of anti-apoptotic mechanisms, such as the deregulation of speci®c signal transduction cascades, the loss of p53 (Kinzler and Vogelstein, 1994) or the activation of protective Bcl-2 family members (Korsmeyer, 1995;Reed, 1996; Kroemer, 1997). Any manipulation that would shift this intricate balance between pro-and anti-apoptotic pathways could have profound e ects on the fate of a tumor cell, either on its own or in combination with conventional anti-cancer drugs. Since the Raf-Ras pathway is activated in many human tumors, we have been interested in studying its role in controlling PCD in tumor cells. This interest was fostered by two recent reports (Monia et al., 1996a, b) which showed that an anti-sense phosphorothioate oligonucleotide (ODN) speci®cally targeted against c-raf mRNA (ISIS5132) had profound e ects on the growth of the human lung adenocarcinoma cell line A549 both in culture and on xenografts in nude mice. The underlying mechanisms remained, however, unclear.In a ®rst set of experiments, we investigated whether the anti-sense ODN ISIS5132 (Monia et al., 1996a) (subsequently referred to as AS) would also reduce the expression of c-Raf protein in, and a ect the growth of, other tumor cell lines. As shown by the immunoblot in Figure 1, treatment of the lung tumor line A549, the cervical carcinoma cell line HeLa, the prostate carcinoma cell line LNCaP or the colon tumor cell line SW620 with AS led to a dramatic reduction of cRaf protein to levels below the detection limit of the assay. In contrast, a mismatched version of this ODN (Monia et al., 1996a) (termed MM in the present study) had no detectable e ect relative to untreated cont...

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Constitutive Raf-1 kinase activity in breast cancer cells induces both estrogen-independent growth and apoptosis

Cited by 96 publications

References 27 publications

Expression of DNA methyl-transferase (DMT) and the cell cycle in human breast cancer cells

Expression of DNA methyl-transferase (DMT) and the cell cycle in human breast cancer cells

Growth of hormone-dependent MCF-7 breast cancer cells is promoted by constitutive caveolin-1 whose expression is lost in an EGF-R-mediated manner during development of tamoxifen resistance

Abrogation of c-Raf expression induces apoptosis in tumor cells

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