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Cancers 2020, 12, 2371 8 of 14 Data are presented as mean ± SD, n = 3 independent experiments and analyzed by ANOVA test. * p < 0.05 for normalized SQSTM1 Bup vs CTL and for relative MAP1LC3B Bup + CQ vs CTL and Omi + CQ vs CTL. (C) Autophagy flux analysis with tandem sensor RFP-GFP-LC3B. Top: GFP is sensitive to acidic pH of AL. Red = AL, yellow = AV. Bottom: Confocal images and AL and AV counts per cell with specialized Image J macro analysis. SCC-9 cells were transduced with the tandem sensor 24 h before 24 h incubation with inhibitors: 1 nM Rapa, 10 μM CQ. Baf (0.3 uM) was added for the last 4 h. The cells were stained with DAPI. PI3Ki concentrations were 0.5 μM Bup and 1 nM Omi. Scale bar = 50 μm. Data are presented as mean ± SD, six images from two independent experiments (three images by experiment per condition with 15–30 cells per picture) and analyzed by unpaired Student’s t-test: *p<0.05forALRapaandALOmivsALCTLand**p<0.01forALBupvsALCTL,*p<0.05 for AV Rapa + Baf and AV CQ vs AV CTL and between AV Omi and AV Omi + CQ, ** p < 0.01 for AV Bup vs AV Bup + C. Uncropped blots are shown in Figure S2. 3. Discussion In this study, we demonstrated that autophagy was activated by two PI3Ki with different mechanisms of action: a pan-class I PI3K family lipid kinase inhibitor, Bup, and a dual PI3K/MTOR inhibitor, Omi. In both cases, CQ prevented the PI3Ki-induced autophagy, as indicated by AV accumulation. We propose that autophagy in HNSCC tumor cells is protective, as tumor cells use autophagy for ROS protection and as an energy source [13,14]. In this context, CQ blocks a tumor escape mechanism to treatment and provides a therapeutic approach that combines CQ with PI3Ki for a better anti-proliferative effect. We used TP53 mutated HPV-negative HNSCC cell lines, which was a limitation in representing the range in heterogeneity of HNSCC found by the TCGA study [3,4]. HPV-positive HNSCC was not evaluated, as it is a very different disease, mostly relevant for oropharyngeal cancer and responds much better to current therapies [49]. Current clinical studies for HPV-positive HNSCC mostly investigate a potential reduction of therapy instead of adding new treatments for patients who usually have a good prognosis. On the other hand, HPV-negative HNSCC therapy is in need of improvement. TP53 status has been shown to influence responses to autophagy inhibition. For example, in TP53 null mice, autophagy inhibition was shown to promote cancer growth [50]. In this context, we focused on TP53 mutated HNSCC which is most frequent among HPV-negative HNSCC. Moreover, a recent computational analysis of the TP53 mutational landscape in HNSCC showed that specific mutations were associated with distinct pathobiological pathways and prognostic signatures [51]. CQ or HCQ have shown limited efficacy as monotherapies in clinical trials. The same is true for PI3K signaling pathway inhibitors. Our results show that the efficacy of combination treatment was not dependent on PIK3CA tumor status. Early studies showed that Bup exhibited preferential inhibition of tumor cells bearing PIK3CA mutations, in contrast to either KRAS or PTEN mutant models [5]. In patients, PIK3CA mutations were neither necessary nor predictive of the response to Omi or Bup treatment (Bup in combination with paclitaxel) [10,11]. Our results are in line with these observations and they indicate that other elements are involved in the cell response to treatment. Recent studies in mutant KRAS- or BRAF-driven mouse models of cancer, such as pancreatic ductal adenocarcinoma cancer (PDAC), demonstrated synergistic antitumor activity with dual autophagy inhibition and mitogen-activated protein kinase kinase (MEK) or mitogen-activated protein kinase (ERK) inhibitors [28,34]. The RAF-MEK-ERK signaling pathway is another major signaling pathway that controls cell proliferation. These results, along with our findings and other studies focusing on PI3K pathway inhibition, suggest that therapeutic strategies using inhibitors of a major cell proliferation signaling pathway, such as PI3K or MAPK, disrupt the signaling cascade and drive cells toward autophagy, enhancing cell responsiveness to autophagy inhibition. This strategy must be adapted to the type of cancer (PI3Ki for HNSCC or MAPK inhibitors for PDAC), and also to the genetic variants/subtypes within each cancer. For example, the TCGA analysis of HNSCC suggests that a small proportion of cases are a MAPK-dependent type of cancer [3]. We anticipate that the development of precision medicine will help to determine the leading proliferative pathway in each individual cancer,PDF Image | Dual Inhibition of Autophagy Pathway as a Therapeutic Strategy
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