Chloroquine sensitizes breast cancer cells to chemotherapy independent of autophagy

Abstract

Chloroquine (CQ) is a 4-aminoquinoline drug used for the treatment of diverse diseases. It inhibits lysosomal acidification and therefore prevents autophagy by blocking autophagosome fusion and degradation. In cancer treatment, CQ is often used in combination with chemotherapeutic drugs and radiation because it has been shown to enhance the efficacy of tumor cell killing. Since CQ and its derivatives are the only inhibitors of autophagy that are available for use in the clinic, multiple ongoing clinical trials are currently using CQ or hydroxychloroquine (HCQ) for this purpose, either alone, or in combination with other anticancer drugs. Here we show that in the mouse breast cancer cell lines, 67NR and 4T1, autophagy is induced by the DNA damaging agent cisplatin or by drugs that selectively target autophagy regulation, the PtdIns3K inhibitor LY294002, and the mTOR inhibitor rapamycin. In combination with these drugs, CQ sensitized to these treatments, though this effect was more evident with LY294002 and rapamycin treatment. Surprisingly, however, in these experiments CQ sensitization occurred independent of autophagy inhibition, since sensitization was not mimicked by Atg12, Beclin 1 knockdown or bafilomycin treatment, and occurred even in the absence of Atg12. We therefore propose that although CQ might be helpful in combination with cancer therapeutic drugs, its sensitizing effects can occur independently of autophagy inhibition. Consequently, this possibility should be considered in the ongoing clinical trials where CQ or HCQ are used in the treatment of cancer, and caution is warranted when CQ treatment is used in cytotoxic assays in autophagy research.

Figure 1

67NR and 4T1 mouse breast cancer cells induce autophagy in response to starvation. 67NR and 4T1 cells were treated with EBSS ± CQ for 2 h to measure autophagy induction with an LC3 western blot (A). 67NR cells were also evaluated for starvation-induced autophagy with fluorescence microscopy using a GFP-cherry-LC3 construct. In (B), 67NR cells were starved for 3 h in EBSS ± 30 µM CQ and observed in a time-lapse confocal microscope. Pictures show frames of each movie at the indicated times (h). Arrows show yellow dots. Graphs in (A) show density analysis of the mean ± standard error of three independent experiments.

Figure 2

Establishment of an inducible system to manipulate autophagy. 67NR and 4T1 cells were transduced with a lentivirus containing either an inducible Atg12 shRNA or a nonsilencing (NS) shRNA (see Materials and Methods). Atg12 was decreased after 72 h of doxycycline (doxy) treatment (A) as well as starvation-induced changes in LC3II (B) and re-distribution of LC3 (C–E). Pictures in (E) show a magnification of CQ and EBSS+CQ pictures in (D). Since doxycycline-treated cells showed less total green fluorescence than non-doxycycline treated cells, pictures in (E) for doxycycline-treated cells (+ doxy) were brightness-enhanced by 20% for a better comparison. Graphs in (B) show density analysis of three independent western blots; graphs in (C) show quantification of green dots from pictures shown in Figure 2D and E and Figure S2. All graphs show mean ± standard error of three independent experiments. Scale bars in (D and E) represent 20 µm.

Figure 3

Chemotherapeutic drugs induce autophagy in the 67NR and 4T1 cell lines. Cells were treated with 1 mM cisplatin, (Cisp) for 6 h or with 30 µM LY294002 (LY) or 0.2 µM rapamycin (Rapa) for 8 h ± CQ and proteins were collected for WB (A). GFP-cherry-LC3 expressing 67NR cells grown in coverslips were treated with the same drug concentrations for 8 h, fixed and observed in a confocal microscope. The bar in merged images represents 20 µM and the one in the inset represents 10 µM. Quantification of yellow dots is shown in (C). Graph shows mean ± standard error of three independent experiments.

Figure 4

CQ sensitizes to LY294002 and rapamycin treatment but has a minimal effect on sensitization to cisplatin treatment. 67NR (A) and 4T1 (B) cells were treated with cisplatin, LY294002 or rapamycin at the indicated doses ± CQ. For short-term (MTS) assays, cells were treated for 24 h with cisplatin or for 48 h with LY294002 or rapamycin (67NR cells) or for 72 h with rapamycin (4T1 cells) with or without 5 mM (CQ 5) or 10 mM CQ (CQ 10). When not indicated, CQ concentration was 10 µM. For long-term (clonogenic) assays, cells were treated for 24 h ± CQ, the treatment was washed and cells were allowed to recover for 5 d. Graphs are normalized to 100% per treatment and show mean ± standard error of three independent experiments done in triplicate. * = statistical difference, p = 0.05.

Figure 5

Autophagy inhibition by Atg12 knockdown has no effect on cell survival in 67NR cells treated with cisplatin, LY294002 or rapamycin. 67NR cells expressing an inducible Atg12 shRNA or a nonsilencing shRNA were treated with doxycycline and then with 1 µM cisplatin, 30 µM LY294002 or 0.2 µM rapamycin (A) or at the indicated concentrations (B and C). In (A), protein was collected for western blot after an 8 h treatment ± CQ. In (B), viability was measured with MTS reagent (short-term assay) after 24 h (cisplatin) or 48 h (LY294002 or rapamycin). In (C), cells grown in the presence of doxycycline were treated for 24 h, the treatment was washed, replaced with fresh medium ± doxycycline (doxy) and cells were allowed to recover for 4 d (long-term, clonogenic assays). Graphs are normalized to 100% per treatment and show mean ± standard error from three independent experiments performed in triplicate.

Figure 6

CQ induced sensitization to LY294002 and rapamycin treatment is not mimicked by Atg12 knockdown and CQ sensitizes even in the absence of Atg12. In (A), 4T1 cells were treated with LY294002 for 48 h or with rapamycin for 72 h ± doxycycline (doxy) or CQ and viability was evaluated in a short-term MTS assay. In (B), cells were grown in the absence or presence of doxycycline for 72 h, treated for 24 h ± doxycycline or CQ, the treatment was washed and replaced with fresh medium ± doxycycline and the cells were allowed to recover for 4 d for long-term (clonogenic) assays. Graphs in (A) show mean ± standard deviation of one representative experiment from three independent experiments performed in triplicate. Graphs in (B) show mean ± standard error of three independent experiments performed in triplicate. In (B), all the treatments were normalized to untreated controls.

Figure 7

Autophagy inhibition with bafilomycin A1 (Baf A) does not have the same effect as CQ. 67NR and 4T1 cells were treated with 20 µM LY294002 (4 h) or 0.2 µM rapamycin (6 h) ± 1 nM Baf A for 4 h and protein was collected for western blot (A). Cells were treated with LY294002 (48 h) or rapamycin (48 h for 67NR and 72 h for 4T1) ± 1 nM Baf A. Viability was measured with MTS reagent for short-term assays (B) and with colony formation assays for long-term assays after a 24 h treatment with the drugs (C). Graphs show mean ± standard deviation of one representative experiment from three independent experiments performed in triplicate.