The small GTPase ADP-Ribosylation Factor 1 mediates the sensitivity of triple negative breast cancer cells to EGFR tyrosine kinase inhibitors

Abstract

The clinical use of EGFR-targeted therapy, in triple negative breast cancer patients, has been limited by the development of resistance to these drugs. Although activated signaling molecules contribute to this process, the molecular mechanisms remain relatively unknown. We have previously reported that the small GTPase ADPRibosylation Factor 1 (ARF1) is highly expressed in invasive breast cancer cells and acts as a molecular switch to activate EGF-mediated responses. In this study, we aimed at defining whether the high expression of ARF1 limits sensitivity of these tumor cells to EGFR inhibitors, such as gefitinib. Here, we show that the knock down of ARF1 expression or activity decreased the dose and latency time required by tyrosine kinase inhibitors to induce cell death. This may be explained by the observation that the depletion of ARF1 suppressed gefitinib-mediated activation of key mediators of survival such as ERK1/2, AKT and Src, while enhancing cascades leading to apoptosis such as the p38MAPK and JNK pathways, modifying the Bax/Bcl2 ratio and cytochrome c release. In addition, inhibiting ARF1 expression and activation also results in an increase in gefitinib-mediated EGFR internalization and degradation further limiting the ability of this receptor to promote its effects. Interestingly, we observed that gefitinib treatment resulted in the enhanced activation of ARF1 by promoting its recruitment to the receptor AXL, an important mediator of EGFR inhibition suggesting that ARF1 may promote its pro-survival effects by coupling to alternative mitogenic receptors in conditions where the EGFR is inhibited. Together our results uncover a new role for ARF1 in mediating the sensitivity to EGFR inhibition and thus suggest that limiting the activation of this GTPase could improve the therapeutic efficacy of EGFR inhibitors.

Functional disruption of the Golgi apparatus protein ARF1 sensitizes MDA-MB-231 breast cancer cells to the antitumor drugs Actinomycin D and Vinblastine through ERK and AKT signaling

“…To further analyze the sensitivity of MDA-MB-231 cells to the combined treatments of ActD or VLB and ARF1 disruptors, we analyzed cell death by apoptosis, by assessing binding of cells to Alexa-Fluor-488-conjugated Annexin V. Both ActD and VLB induce cell death by apoptosis [ 94 , 95 ], and accordingly we found that both significantly increased the apoptosis of MDA-MB-231 cells ( Fig 5 ). We also found that the treatment with each of the ARF1 disruptors significantly increased the apoptosis of MDA-MB-231 cells ( Fig 5 , and data not shown), in agreement with previous reports [ 69 , 96 ]. Importantly, the combined treatments also resulted in significant increases in apoptosis, but to a higher extent than in single ARF1 disruptor treatments, or single antitumor drug treatments ( Fig 5 ).…”

“…Similarly, the PI3K/AKT signaling pathway plays important roles in both estrogen receptor negative and estrogen receptor positive breast tumor cells [ 97 , 98 ]. In fact, both pathways seem to mediate several tumorigenic responses in MDA-MB-231 cells [ 99 ], and in an ARF1-dependent manner [ 96 ]. Thus, to explore the mechanism of sensitization of MDA-MB-231 cells to ActD and VLB, we analyzed the ERK1/2 and AKT signaling pathways evaluating the levels of phospho-ERK1/2 and phospho-AKT by immunoblot.…”

ANXA2 could act as a moderator of EGFR-directed therapy resistance in triple negative breast cancer

“…Gefitinib was thought to inhibit cellular proliferation and tumour growth in human non-small cell cancer (NSCLC) dose-dependently [22], and potentiated the cytotoxic effects of chemotherapy and/or radiation. Previous study set the gefitinib dose as geometric progression (0 µM, 0.1 µM, 1 µM, 10 µM, 100 µM) to investigate the IC50 value of breast cancer cell lines [35]. In the present research, to explore the viability of cells at higher concentration, we set series concentration of gefitinib (6.25 µM, 12.5 µM, 25 µM, 50 µM, 100 µM, 200 µM).…”

Design and Synthesis of Arf1-Targeting γ-Dipeptides as Potential Agents against Head and Neck Squamous Cell Carcinoma

“…Notably, Arf1 has been reported to be activated downstream of the EGFR in triple negative breast cancer cells (TNBC) cells [43]. Since then, several reports have underlined the importance of the Arf1/EGFR axis in cancer cells progression [46][47][48]. In HNSCC, we have previously evidenced direct interaction between Arf1 and phospho-EGFR and highlighted the critical role of the EGFR-Arf1 complex in driving HNSCC progression [14].…”