Inhibition of autophagy by chloroquine prevents resistance to PI3K/AKT inhibitors and potentiates their antitumor effect in combination with paclitaxel in triple negative breast cancer models

Abstract

Background: Triple negative breast cancer (TNBC) is an aggressive disease characterized by high risk of relapse and development of resistance to different chemotherapy agents. Several targeted therapies have been investigated in TNBC with modest results in clinical trials. Among these, PI3K/AKT inhibitors have been evaluated in addition to standard therapies, yielding conflicting results and making attempts on elucidating inherent mechanisms of resistance of great interest. Increasing evidences suggest that PI3K/AKT inhibitors can induce autophagy in different cancers. Autophagy represents a supposed mechanism of drug-resistance in aggressive tumors, like TNBC. We, therefore, investigated if two PI3K/AKT inhibitors, ipatasertib and taselisib, could induce autophagy in breast cancer models, and whether chloroquine (CQ), a well known autophagy inhibitor, could potentiate ipatasertib and taselisib anti-cancer effect in combination with conventional chemotherapy.

Methods: The induction of autophagy after ipatasertib and taselisib treatment was evaluated in MDAMB231, MDAM468, MCF7, SKBR3 and MDAB361 breast cancer cell lines by assaying LC3-I conversion to LC3-II through immunoblotting and immunofluorescence. Other autophagy-markers as p62/SQSTM1 and ATG5 were evaluated by immunoblotting. Synergistic antiproliferative effect of double and triple combinations of ipatasertib/taselisib plus CQ and/or paclitaxel were evaluated by SRB assay and clonogenic assay. Anti-apoptotic effect of double combination of ipatasertib/taselisib plus CQ was evaluated by increased cleaved-PARP by immunoblot and by Annexin V- flow cytometric analysis. In vivo experiments were performed on xenograft model of MDAMB231 in NOD/SCID mice.

Results: Our results suggested that ipatasertib and taselisib induce increased autophagy signaling in different breast cancer models. This effect was particularly evident in PI3K/AKT resistant TNBC cells, where the inhibition of autophagy by CQ potentiates the therapeutic effect of PI3K/AKT inhibitors in vitro and in vivo TNBC models, synergizing with taxane-based chemotherapy.

Conclusion: These data suggest that inhibition of authophagy with CQ could overcome mechanism of drug resistance to PI3K/AKT inhibitors plus paclitaxel in TNBC making the evaluation of such combinations in clinical trials warranted.

Keywords: Autophagy; Breast Cancer; Chloroquine; PI3K/AKT/mTOR inhibitors; TNBC.

Fig. 1

Ipatasertib and taselisib reduce cell proliferation in breast cancer models. a and ability to prevent clonogenic formation after daily administration for 10 days of IC30 doses for each drug or at fixed drug dose of 1 µM, expressed as % of CTL. Each experiment is representative of three independent experiments. b Ipatasertib and taselisib treatment impair 3D tumor spheroid derived from breast cancer cells. Representative images of first generation 3D tumor spheroid, exposed to ipatasertib and taselisib, administrated to IC30 doses for 72 h. Tumor cell growth was reduced in MCF7 and SKBR3 while MDAMB231 and MDAMB468 cells form only aggregates. Quantification of ATP was used to measure reduction of cellular growth, expressed as % of CTL. Each experiment is representative of three independent experiments. Statistically significant results are reported (*** indicates P < 0.0005, ** indicates P < 0.005 and * indicates P < 0.05)

Fig. 2

Ipatasertib and taselisib treatment induces Autophagy in all breast cancer analyzed. a The exposure to fixed dose (IC50) of ipatasertib and taselisib induces the reduction of expression of phospho-mTOR, associated with the increase of autophagy signaling, as showed by increase of LC3 II/LC3 I ratio by immunoblot assay by reduction of p62 and p-ULK1-ser758, or increase of ATG5 after 24 h of exposure in MCF7, SKBR3, MDAMB231 and MDAMB468 cell lines. In MDAMB231 cell line the maximum dosage of 10 µM was used for both drugs. Statistically significant results are reported (*** indicates P < 0.0005, ** indicates P < 0.005 and * indicates P < 0.05)

Fig. 3

Chloroquine reduces autophagic flux and induce apoptosis in TNBC cell lines. a The addition of CQ 10 µM to ipatasertib and taselisib (IC50) induces accumulation of LC3II and p62 protein expression after 24 h, due the reduction of autophagic flux, while expression of cleaved parp was increased in taselisib + CQ group in MDAMB231 cell line and in ipatasertib + CQ group in MDAMB468 cell line. b Representative confocal images of cell lines immuno-stained with anti-LC3IIb antibody reveals accumulation of autophagosomes (green dots) in treatments with ipatasertib, taselisib, CQ or combinations, due to the induction of autophagy or reduction of autophagic flux exerted by CQ. In MDAMB231 cell line the maximum dosage of 10 µM was used for both drugs. Statistically significant results are reported (*** indicates P < 0.0005, ** indicates P < 0.005 and * indicates P < 0.05)

Fig. 4

Chloroquine potentiates the antitumor effect of ipatasertib and taselisib in TNBC cells a-d The addition of CQ 1-10 µM to ipatasertib and taselisib inhibits cell proliferation in MDAMB231 and MDAMB468 cell lines, measured by SRB assay after 72 h and expressed as % of CTL. Each experiment is representative of three independent experiments. b By citofluorimetric assay, CQ 10 µM in combination with taselisib 10 µM, increases apoptosis, as measured by enhancement of Annexin V-FITC and Propidium Iodide, after 24 of treatment in MDAMB231, expressed as % of CTL e CQ alone or in combination with ipatasertib 1 µM, increases apoptosis, as measured by enhancement of Annexin V-FITC and Propidium Iodide, after 48 h of treatment in MDAMB468, expressed as % of CTL. Each experiment is representative of three independent experiments. c Daily exposure for 10 days of low dose (58 nM) of CQ reduces clonogenic proliferation of MDAMB231 cell line alone or in combination with ipatasertib and taselisib (1 µM). Cell growth was represented as % of CTL. f Daily exposure for 10 days of low dose (1 µM) of CQ reduces clonogenic proliferation of MDAMB468 cell line in combination with ipatasertib and taselisib (1 µM). Cell growth was represented as % of CTL. Each experiment is representative of three independent experiments. Statistically significant results are reported (*** indicates P < 0.0005, ** indicates P < 0.005 and * indicates P < 0.05)

Fig. 5

Chloroquine potentiates the antitumoral activity of chemotherapy and PI3K/AKT inhibitors combination in TNBC cell lines a-c Daily exposure for 10 days to triple combination of low doses of CQ (58 nM-1 µM), paclitaxel (0,5 nM), ipatasertib (1 µM) or taselisib (1 µM) exposure, prevent clonogenic formation in MDAMB231 and MDAMB468 cell lines. Representative experiments, similar experiments yeld similar results b-d Triple combination of low doses of CQ (1 µM), paclitaxel (1 nM), ipatasertib (1–10 µM) or taselisib exposure, to the indicated treatments, induces the inhibition of cell proliferation in MDAMB231 and MDAMB468 cell lines, measured by SRB assay after 72 h and expressed as % of CTL. Each experiment is representative of three independent experiments. Statistically significant results are reported (*** indicates P < 0.0005, ** indicates P < 0.005 and * indicates P < 0.05)

Fig. 6

Combination of CQ and taselisib potentiates paclitaxel antitumor effect in vivo TNBC xenograft models. a MDAMB231 cells (7 × 10⁶) were s.c. injiected in NOD/SCID mice as described in Methods section. When established tumors were palpable, mice were treated with 1) vehicles (CTL group) 2)taselisib (5 mg/kg/os) daily 3)CQ (30 mg/kg/os) daily 4)paclitaxel (10 mg/Kg/IP) weekly 5)taselisib + CQ daily and 6)taselisib + paclitaxel 7)paclitaxel + CQ 8)triple combination. Treatment lasted 2 weeks followed by 1 week of follow-up. Relative tumor volume curves are represented as means ± SE measured at pre-specified time points. Inset, body weight have been measured two times/week. Statistically significant results are reported (*** indicates P < 0.0005, ** indicates P < 0.005 and * indicates P < 0.05). b Tumour volume averages from each group at day 0 and day 22 were compared and presented as percentages of vehicle. c Tumor growth delay (TGD) determined as %TGD = [(T − C) /C] × 100, where T and C are the mean times expressed in days for the treated or control groups, respectively, to reach a defined tumor volume (see Materials and Methods). d Representative images of tumors collected ex vivo on day 28 e Tumor volume ex vivo on day 28 represented as means ± SE (f) Tumor weight ex vivo on day 28 represented as means ± SE