Decreased Interaction of Raf-1 with Its Negative Regulator Spry2 as a Mechanism for Acquired Drug Resistance

Biomolecules & Therapeutics

2015

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The clinical benefits of oncogenic BRAF inhibitor therapies are limited by the emergence of drug resistance. In this study, we investigated the role of a negative regulator of the MAPK pathway, Spry2, in acquired resistance using BRAF inhibitor-resistant derivatives of the BRAF-V600E melanoma (A375P/Mdr). Real-time RT-PCR analysis indicated that the expression of Spry2 was higher in A375P cells harboring the BRAF V600E mutation compared with wild-type BRAF-bearing cells (SK-MEL-2) that are resistant to BRAF inhibitors. This result suggests the ability of BRAF V600E to evade feedback suppression in cell lines with BRAF V600E mutations despite high Spry2 expression. Most interestingly, Spry2 exhibited strongly reduced expression in A375P/Mdr cells with acquired resistance to BRAF inhibitors. Furthermore, the overexpression of Spry2 partially restored sensitivity to the BRAF inhibitor PLX4720 in two BRAF inhibitor-resistant cells, indicating a positive role for Spry2 in the growth inhibition induced by BRAF inhibitors. On the other hand, long-term treatment with PLX4720 induced pERK reactivation following BRAF inhibition in A375P cells, indicating that negative feedback including Spry2 may be bypassed in BRAF mutant melanoma cells. In addition, the siRNA-mediated knockdown of Raf-1 attenuated the rebound activation of ERK stimulated by PLX4720 in A375P cells, strongly suggesting the positive role of Raf-1 kinase in ERK activation in response to BRAF inhibition. Taken together, these data suggest that RAF signaling may be released from negative feedback inhibition through interacting with Spry2, leading to ERK rebound and, consequently, the induction of acquired resistance to BRAF inhibitors.

Section: Introductionsupporting

confidence: 68%

Induction of Resistance to BRAF Inhibitor Is Associated with the Inability of Spry2 to Inhibit BRAF-V600E Activity in BRAF Mutant Cells

Ahn

Han

Biomolecules & Therapeutics

2015

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“…Although MDRtargeted therapy is a specific and valid strategy, many MDR modulators are not as effective as expected (de Grouw et al ., 2006), possibly because many tumors acquire drug resistance via a variety of mechanisms besides P-glycoprotein-mediated drug efflux. We recently developed a paclitaxel-resistant Ras- NIH 3T3/Mdr cell line that has a multidrug-resistance phenotype mediated by the P-glycoprotein (Ahn et al ., 2011). In a recent study, we showed that Ras-NIH 3T3/Mdr cells were more susceptible than Ras-NIH 3T3 cells to Src inhibition with PP2, which acts by an MDR-independent mechanism (Ahn and Lee, 2011).…”

Section: Discussionmentioning

confidence: 99%

“…Ras-NIH 3T3 cells contain morphologically transformed foci of cells that exhibit crisscrossed margins, piling-up properties and characteristics of invasiveness (Lee et al ., 2009), and their drug-resistant counterparts (Ras-NIH 3T3/Mdr cells) stably express the drug effl ux pump P-glycoprotein, which can be blocked by verapamil (Ahn et al ., 2011). Both cell lines were maintained at 37℃ in DMEM supplemented with 10% FCS, penicillin-streptomycin, and glutamine.…”

Section: Methodsmentioning

confidence: 99%

Src Family Kinase Inhibitor PP2 Induces LC3 Conversion in a Manner That is Uncoupled from Autophagy and Increases Apoptosis in Multidrug-Resistant Cells

Kim

Ahn²,

Biomolecules and Therapeutics

2012

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Recently, we reported that defective autophagy may contribute to the inhibition of the growth in response to PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), a selective SFK inhibitor, in multidrug-resistant v-Ha-ras-transformed NIH 3T3 cells (Ras-NIH 3T3/Mdr). In this study, we demonstrated that PP2 induces LC3 conversion via a mechanism that is uncoupled from autophagy and increases apoptosis in Ras-NIH 3T3/Mdr cells. PP2 preferentially induced autophagy in Ras-NIH 3T3 cells rather than in Ras-NIH 3T3/Mdr cells as determined by LC3-I to LC3-II conversion and GFP-LC3 fluorescence microscopy. Beclin 1 knockdown experiments showed that, regardless of drug resistance, PP2 induces autophagy via a Beclin 1-dependent mechanism. PP2 induced a conformational change in Beclin 1, resulting in the enhancement of the pro-autophagic activity of Beclin 1, in Ras-NIH 3T3 cells. Further, PI3K inhibition induced by wortmannin caused a significant increase in apoptosis in Ras-NIH 3T3 cells, as demonstrated by flow cytometric analysis of Annexin V staining, implying that autophagy inhibition through PI3K increases apoptosis in response to PP2 in Ras-NIH 3T3 cells. However, despite the fact that wortmannin abrogates PP2-induced GFP-LC3 punctae formation, some LC3 conversion remains in Ras-NIH 3T3/Mdr cells, suggesting that LC3 conversion may occur in an autophagy-independent manner. Taken together, these results suggest that PP2 induces LC3 conversion independent of PI3K, concomitant with the uncoupling of LC3 conversion from autophagy, in multidrug-resistant cells.

“…Our previous study demonstrated that the Ras‐NIH 3T3/Mdr cell line stably expresses the drug efflux pump P‐glycoprotein that can be blocked by verapamil (Ahn et al, 2011). We first evaluated the expression of mdr‐1 and mdr‐3 in Ras‐NIH 3T3/Mdr cells with real‐time PCR analysis (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The Ras‐NIH 3T3 cells grow as morphologically transformed foci with the characteristics of crisscrossed margins, the piling up of cells and invasiveness (Lee et al, 2009). The development of multidrug‐resistant Ras‐NIH 3T3/Mdr cells, which express large amounts of P‐glycoprotein compared with their parental counterparts, has been previously described (Ahn et al, 2011). The Ras‐NIH 3T3/Mdr cells and their parental counterparts were maintained at 37°C in DMEM supplemented with 10% FCS, penicillin‐streptomycin, and glutamine.…”

Section: Methodsmentioning

confidence: 99%

Defective autophagy in multidrug resistant cells may lead to growth inhibition by BH3‐mimetic gossypol

Ahn

Jang

Journal Cellular Physiology

2013

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The clinical efficacy of many chemotherapeutic agents has been reduced due to the development of drug resistance. In this article, we aimed to validate gossypol, a natural BH3 mimetic found in cottonseeds, as a potential therapeutic to overcome multidrug resistance (MDR). Gossypol was found to retain its efficacy in v-Ha-ras-transformed NIH 3T3 cells that overexpressed P-glycoprotein (Ras-NIH 3T3/Mdr), which was similar to the efficacy observed in their parental counterparts (Ras-NIH 3T3). A rhodamine assay revealed that the alteration of MDR activity did not contribute to the cytotoxic effect of gossypol. Gossypol caused a G2 /M arrest by the induction of p21(Cip1) and the down-regulation of p27(Kip1) expression in Ras-NIH 3T3 cells, whereas no significant G2 /M arrest was exhibited in Ras-NIH 3T3/Mdr cells. Surprisingly, a 48-h treatment with gossypol induced apoptotic cell death in Ras-NIH 3T3 cells; however, gossypol induced both apoptosis and necrosis in Ras-NIH 3T3/Mdr cells, as determined with flow cytometry analysis. More notably, gossypol preferentially induced autophagy in Ras-NIH 3T3 cells but not in Ras-NIH 3T3/Mdr cells. Coimmunoprecipitation and flow cytometric analysis revealed that gossypol-induced autophagy is independent of the dissociation of Beclin 1 from Bcl-2 in Ras-NIH 3T3 cells. Taken together, these results suggest that the antiproliferative activity of gossypol appears to be due to cell-cycle arrest at the G2 /M phase, with the induction of apoptosis in Ras-NIH 3T3 cells. In addition, defective autophagy might contribute to apoptotic and necrotic cell death in response to gossypol in Ras-NIH 3T3/Mdr cells.