Combined SFK/MEK inhibition prevents metastatic outgrowth of dormant tumor cells
Lara H. El Touny, Anthony Vieira, Arnulfo Mendoza, Chand Khanna, Mark J. Hoenerhoff, Jeffrey E. Green
Lara H. El Touny, Anthony Vieira, Arnulfo Mendoza, Chand Khanna, Mark J. Hoenerhoff, Jeffrey E. Green
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Research Article Oncology

Combined SFK/MEK inhibition prevents metastatic outgrowth of dormant tumor cells

Abstract

Breast cancer (BC) can recur as metastatic disease many years after primary tumor removal, suggesting that disseminated tumor cells survive for extended periods in a dormant state that is refractory to conventional therapies. We have previously shown that altering the tumor microenvironment through fibrosis with collagen and fibronectin deposition can trigger tumor cells to switch from a dormant to a proliferative state. Here, we used an in vivo preclinical model and a 3D in vitro model of dormancy to evaluate the role of Src family kinase (SFK) in regulating this dormant-to-proliferative switch. We found that pharmacological inhibition of SFK signaling or Src knockdown results in the nuclear localization of cyclin-dependent kinase inhibitor p27 and prevents the proliferative outbreak of dormant BC cells and metastatic lesion formation; however, SFK inhibition did not kill dormant cells. Dormant cell proliferation also required ERK1/2 activation. Combination treatment of cells undergoing the dormant-to-proliferative switch with the Src inhibitor (AZD0530) and MEK1/2 inhibitor (AZD6244) induced apoptosis in a large fraction of the dormant cells and delayed metastatic outgrowth, neither of which was observed with either inhibitor alone. Thus, targeting Src prevents the proliferative response of dormant cells to external stimuli, but requires MEK1/2 inhibition to suppress their survival. These data indicate that treatments targeting Src in combination with MEK1/2 may prevent BC recurrence.

Authors

Lara H. El Touny, Anthony Vieira, Arnulfo Mendoza, Chand Khanna, Mark J. Hoenerhoff, Jeffrey E. Green

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Figure 7

Combination of AZD0530 and AZD6244 induces apoptosis of D2.0R cells on BME plus COL1 and dramatically reduces their metastatic burden in fibrotic lungs.

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Combination of AZD0530 and AZD6244 induces apoptosis of D2.0R cells on B...
(A) pERK1/2 levels in D2.0R cells treated with 250 nM AZD0530 or vehicle for 72 hours. (B) Caspase 3/7 assays of D2.0R cells treated with AZD0530 (250 nM), AZD6244 (500 nM), or both with or without Z-VAD-FMK for 48 hours. **P = 0.0034; ***P = 0.0011. (C) Annexin V/PI staining of D2.0R cells treated with vehicle, AZD0530, AZD6244, or their combination, as above, for 48 hours with quantification ± SEM from three experiments. (D) MTS assay for D2.0R seeded on BME plus COL1 and treated, as above, for 96 hours. *P = 0.0051, **P = 0.0029, and ***P = 0.0007 compared with vehicle-treated cells; also, P = 0.0061, P = 0.0194, and P = 0.0025 comparing AZD0530 versus AZD6244, AZD0530 versus combination, and AD6244 versus combination, respectively. (E) MTS values of similarly treated D2.0R cells with or without Z-VAD-FMK at 48 hours. *P = 0.0282, **P = 0.0016, and ***P = 0.0013 compared with control vehicle–treated D2.0R cells. (F) Lung metastatic burden represented in pixels2 of CD1nu/nu mice instilled with Ad–TGF-β223/225, then tail-vein injected with 1 × 106 D2.0R GFP cells, followed by gavage 24 hours later with vehicle, AZD0530, AZD6244, or their combination once daily for 21 days.