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Cancers 2020, 12, 2371 5 of 14 tissues to toxicity levels observed with long-term high dosages used for malaria treatment such that CQ has been associated with retinopathy [30]. Although CQ has several dose-dependent effects, cancer patients could benefited from CQ treatment at a dose of 31 μM (10 mg/kg CQ-Base or 16.7 mg/kg CQ-diphosphate, daily) [39]. We used low concentrations for each inhibitor to measure any enhanced decrease in cell proliferation when inhibitors were used in combination. For CQ, we used a range of concentrations, from 1 to 30 μM. We observed a decrease in the proliferation of SCC-9 and FaDu cells in response to CQ or PI3Ki treatment (Figure 2A and Figure S1A). The effect of PI3Ki on proliferation was significantly enhanced when CQ was added to the treatment. We performed the same experiment with a panel of HPV-negative HNSCC cell lines harboring the most common PIK3CA alterations (Figure 2D), but all mutated for TP53 [40–44]. TP53 mutated cell lines represent 86% of HPV-negative HNSCC cases [3], and a study found TP53 mutations in 95% of metastatic HNSCC [45]. Real-time cell proliferation assays for this panel showed the same phenomenon: population doubling (PD) was significantly decreased with the combination of CQ and PI3Ki in five of six HNSCC cell lines (SCC-4 not shown, Figure 2B). SCC-9 dose-response curves for each inhibitor, alone or in combination, were analyzed while using the SynergyFinder software [46] with the Bliss independence model [47,48] showing high synergy between PI3Ki Bub or Omi and CQ (Figure 2C). Bliss synergy analysis for the other HNSCC cell lines of the panel (Figure S1B) demonstrated a high synergy between PI3Ki and CQ, as summarized in Figure 2D, with similar trends in cell line sensitivity for Bup and Omi and appeared independent of PIK3CA mutation/amplification status. Our results advocate for the use of a combination therapy in HNSCC, which would include CQ in addition to PI3Ki. 2.3. CQ Inhibits PI3Ki-Induced Autophagy Our results suggest that the induction of autophagy associated with the concomitant inactivation of the PI3K pathway is blocked by CQ. To verify this, SCC-9 cells were incubated for one day with Omi, CQ, or the combination of both and stained with acridine orange. Incubation with PI3Ki Omi increased the number of acidic organelles in the HNSCC cell lines due to autophagy activation (Figure 3A). We observed an increase in acidic organelle content with CQ, which inhibits autophagy at a late stage: the fusion of AV to lysosomes is blocked leading to AV accumulation. We observed a further increase of acidic organelles with the combination, suggesting the accumulation of acidic organelles caused by an inhibition of PI3Ki-induced autophagy. We made the same observation when FaDu cells were exposed for one day to Bup, CQ, or both inhibitors (Figure S1C). We examined two markers of autophagy previously described to analyze the role of each pathway in the combination setting: MAP1LC3BII and SQSTM1. We observed a decrease of SQSTM1 and an increase of MAP1LC3BII levels in the presence of PI3Ki. With CQ, SQSTM1 degradation was prevented and MAP1LC3BII accumulated in the cells. The MAP1LC3BII levels were further increased by the combination treatments, showing that CQ blocked autophagy induced by PI3K inhibition (Figure 3B). We also analyzed the autophagic flux with the tandem RFP-GFP-LC3B sensor (Figure 3C top). AVs have been shown to accumulate following the addition of drugs, such as CQ and the autophagy inhibitor Bafilomycine A1 (Baf), which blocks autophagy through lysosomal pH neutralization and prevents fusion with lysosomes [15,28,34]. The number of red LC3B vesicles increased with rapamycin (Rapa), an MTOR inhibitor that is known to induce autophagy [15], and with PI3Ki, Bup, or Omi, as shown previously. We observed AV (yellow vesicles) accumulation with Rapa + Baf incubation as well as with CQ. AV number by cell was also significantly increased by treatment combinations of CQ and PI3Ki (Figure 3C bottom), demonstrating that CQ inhibits PI3Ki-induced autophagy.PDF Image | Dual Inhibition of Autophagy Pathway as a Therapeutic Strategy
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